Fluorescence energy transfer and intramolecular energy transfer in particles using novel compounds

ABSTRACT

Particles comprising an energy donor as a first component and a fluorescent dye as a second component positioned in said particles at an energy exchanging distance from one another, wherein the two components have a Stokes shift of greater than or equal to 50 nm, said particle having bound on its surface, a protein, polypeptide, nucleic acid, nucleotide or protein containing ligand analogue are disclosed and claimed. In addition, novel fluorescent dyes are described which exhibit intramolecular energy transfer for use to label various molecules, proteins, polypeptides, nucleotides and nucleic acids or to incorporate into particles.

This application is a continuation in part of application Ser. No.08/274,534 filed Jul. 12, 1994 and of application Ser. No. 08/138,708filed Oct. 18, 1993 now abandoned, of application Ser. No. 08/126,367filed Sep. 24, 1993 now abandoned from which priority is claimed. All ofthese applications are hereby fully incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates generally to the synthesis of novel dyes andlabels and methods for the detection or visualization of analytes andmore specifically to fluorescent latex particles which incorporate thenovel fluorescent dyes and utilize, in certain aspects, fluorescenceenergy transfer and intramolecular energy transfer, for the detection ofanalytes in immunoassays or in nucleic acid assays.

BACKGROUND

Various methodologies are available for the visualization of cells ormolecules in cells and for the measurement of analyte concentrations influids. Fluorescence microscopy utilizes fluorescent dyes, generallyconnected to specific probes, such as antibodies, for the localizationof proteins and complexes in cells. For the measurement of analyteconcentrations, immunoassays have become popular over the last 40 yearsbecause of the specificity of antibodies toward the analyte or targetligand. Radioimmunoassays were developed because the high specificactivity of the radionuclide allowed measurement of very lowconcentrations of analyte. However, because of the concerns for theenvironment and human health, the use of radionuclides in immunoassaysis becoming less popular. The use of enzymes in immunoassays to amplifya signal has been a very important advance in the field of immunoassaysbecause their use does not involve environmental or human health hazardsor risks. Enzyme-linked immunoassays, however, can be problematicbecause the activity of the enzyme is temperature dependent and theinstability of the enzyme or the substrates can result in inaccuratequantitation of the target ligand. Still other immunoassays monitorfluorescence as the signal, with or without enzymes, for the measurementof analyte concentrations.

The characteristics of the fluorescent dyes are very important whenquantifying analyte concentrations in biological fluids. For example,when the biological fluid is blood, serum or plasma, the intrinsicfluorescence of the fluid precludes the use of many dyes. Thesebiological fluids generally have fluorescence emissions up to 600 nmwhen exciting at various wavelengths above 200 nm. The fluorescence isgenerated by excitation of the dye at the appropriate wavelength. Thefluorescent signal is measured by a fluorometer which is tuned to excitethe fluorescent molecule at a specific wavelength and to measure theemission of fluorescence at another wavelength. The difference in theexcitation and emission wavelengths is referred to as the Stokes shift.To achieve the most sensitive measurement, the emission wavelength ofthe sample should not interfere with the emission of the dye. Also, theStokes shift should be as large as possible so that the excitation lightis not seen by the detector as a background signal. When the Stokesshift is not large, filters or monochromators can be utilized in thefluorometer to exclude light near the emission wavelength; however, theuse of filters decreases the yield of light reaching the detector andgenerally one circumvents this problem of light loss by the use of highintensity lamps. Thus, to avoid problems associated with small Stokesshifts and dyes which emit near the intrinsic emission of the biologicalfluid, a sophisticated instrument is generally built. With the advent ofnear-patient diagnostics in hospitals, instruments which are used forthe diagnostics will become more portable and simpler to use. Therefore,there is a need for portable, simple fluorometers which can assessfluorescence in an immunoassay for the detection of analytes inbiological samples.

Another problem associated with the assay of analytes in fluids or thevisualization of cellular components with an intrinsic fluorescence isthat of selection of the dye which is utilized as the label. The dye isgenerally chosen for its brightness (the product of fluorescence quantumyield and extinction coefficient) since a certain sensitivity in theassay or the visualization technique is required. However, the selectionof the dye used as the label is limited when the sample has an intrinsicfluorescence because the instrument may not be capable of distinguishingsample fluorescence from dye fluorescence.

The current invention provides a methodology for the development ofamplified fluorescent label systems which can be tuned to specificexcitation and emission wavelengths. In addition, the methodologyteaches improved methods for incorporation of dyes into particles tominimize fluorescence quenching and to maximize fluorescence intensitiesof the dye molecules in the particles. The novel dye systems can beutilized for the quantitation of analytes in fluids, and in particular,in biological fluids. The novel dye systems can be tuned to specificexciting and emitting wavelengths so that low current sources, such aslight emitting diodes and laser diodes, and detectors, such as photodiodes, and the like, can be used in the manufacture of fluorometerswhich can be battery powered and portable, for use, for example, inimmunoassays dedicated to near-patient diagnostics.

SUMMARY OF THE INVENTION

This invention relates to novel fluorescent particles. These novelparticles can be tuned to specific excitation and emission wavelengthsto accommodate a wide variety of assay or visualization systems. In yetanother aspect of the invention, the methodology teaches improvedmethods for incorporation of dyes into particles to minimizefluorescence quenching and to maximize fluorescence intensities of thedye molecules in the particles through the use of different dyemolecules which possess the same or very similar excitation and emissionwavelengths.

Many novel phthalocyanine derivatives and hybrid phalocyaninederivatives are disclosed and claimed. In one embodiment microparticleshaving at least one hybrid phthalocyanine derivative, said derivative(s)having (1) at least one donor subunit with a desired excitation peak;and (2) at least one acceptor subunit with a desired emission peak,wherein said derivative(s) is/are capable of intramolecular energytransfer from said donor subunit to said acceptor subunit are disclosed.Such derivatives also may contain an electron transfer subunit. Axialligands may be covalently bound to the metals contained in the hybridphthalocyanine derivatives. Numerous compounds capable of intramolecularenergy transfer as well as compounds for fluorescence energy transferare claimed.

DESCRIPTION OF THE DRAWING

FIG. 1 depicts the structures of phthalocyanine, naphthalocyanine andanthranylocyanine.

FIG. 2 depicts the structures of silicon phthalocyanine, siliconnaphthalocyanine and silicon anthranylocyanine.

FIG. 3 depicts the spectra of silicon phthalocyanine dihydroxide and thespectra of silicon 2,3-naphthalocyanine dihydroxide.

FIG. 4 depicts the general structure of ethenyl-substituteddipyrrometheneboron difluoro dyes.

FIG. 5 depicts the attenuation of the background signal as a function ofincreasing wavelength. The data was measured using a device as describedin Applicant's allowed Ser. No. 07/887,526 filed May 21, 1992 entitled"Diagnostic Devices and Apparatus for the Controlled Movements ofReagents Without Membranes," now U.S. Pat. No. 5,458,852 which is herebyfully incorporated herein.

FIG. 6 depicts naphthalocyanine derivatives which emit in the nearinfrared.

FIG. 7 depicts general structures of fluorescent energy transfernaphthalocyanine compounds.

FIG. 8 depicts the absorbance spectrum of human serum between 200 nm and1000 nm.

FIG. 9 depicts the structure of a novel hybrid phthalocyaninederivative, Silicon di(1,6-diphenylnaphthalocyanine)! diphthalo-cyaninebis(dimethylhexylvinylsilyloxide).

DETAILED DESCRIPTION

This invention describes novel fluorescent particles and novelfluorescent molecules and diagnostic methods for their use. Developing amethod for the visualization of a cellular component or a cell or for anassay which utilizes a fluorescent dye and which quantifies an analytein a sample requires the use of a fluorometer. The fluorescent label,the sample and the instrument must be compatible with each other toachieve an accurate measurement. Several criteria for a fluorescentlabel as it relates to the sample and instrument are described below.First, the absorption or excitation and emission wavelengths of the dyeshould not correspond to those of the specimen or sample. Second, theStokes shift of the dye should be as large as possible to minimize themeasurement of background from the excitation wavelength. Third, the dyemust be compatible with the phase of the visualization or the fluidphase of the assay; that is, the dye must be water soluble or waterinsoluble depending on the visualization or assay format. Fourth, thedye should be as bright as is necessary to achieve the desiredsensitivity. Brightness is the product of the extinction coefficient andthe quantum yield of the dye. Fifth, the instrument used to detect thefluorescent signal is generally designed around the specifications ofthe dye and the specimen or sample being visualized or assayed.

These points will be discussed in more detail and illustrate some of thedifficulties in developing a fluorescent visualization technique or anassay using fluorescent dyes. One is limited either to dyes which havebeen synthesized or ones which must be synthesized in order to meet theabove criteria. Those skilled in the art will appreciate that the designand synthesis of dye molecules which have a very broad range ofexcitation and emission wavelengths is very tedious and generally, onlya very limited range of excitation and emission wavelengths can beplanned for a specific molecule. The teachings of this invention allowone to prepare fluorescent labels which can be tuned to many excitationand emission wavelengths allowing for large Stokes shifts. Thus,designing a dye system with the specifications of the sample or specimenand the instrument is possible rather than designing the instrumentaround the specifications of the dye. Tuning the dye system toaccommodate the characteristics of the sample and the instrument resultsin a much greater chance of success of the visualization process or theassay.

The excitation and emission wavelengths of the dye should not correspondto those of the sample being assayed or visualized, otherwise the samplecan interfere with the measurement of the fluorescent signal. Whenabsorption or emission wavelengths of the sample do correspond to thoseof the dye, in practice, one dilutes, for example, a serum or bloodsample so that the interference by the sample is reduced or theinterfering sample is washed away from the detection area. Indeed,currently on the market, no fluorescent assay system exists for themeasurement of analytes in neat biological fluids, particularly blood orserum. One reason for the lack of fluorescent assay systems which detectanalytes in neat samples is that no good fluorescent dye exists whichmeets all the criteria listed above, particularly for measuringfluorescence in biological samples. When the sample absorbssignificantly at the excitation wavelength the amount of light whichexcites the sample is thus affected by the variation in the samplecharacteristics. For example, serum, plasma, or blood from differentindividuals will be different in their relative absorptivities, whichwill translate into different intensities of excitation light used toexcite the fluorescent label. The fluorescence emission of the dye isdirectly proportional to the intensity of the incident light, such thatwhen the sample absorbs a portion of the incident light, the intensityof the fluorescent signal will vary accordingly. This results inmeasuring an incorrect or effected fluorescence emission. In addition,the emission wavelength of the dye should not correlate with theemission or absorbance of the sample because the sample will increasethe measured fluorescence of the dye or the sample will absorb all or aportion of the dye fluorescence and also result in an incorrect oreffected fluorescence emission. These problems are avoided when thesample is invisible to the excitation and emission wavelengths.

FIG. 8 shows the spectrum between 200 nm and 1000 nm of human serum.Wavelengths above 600 nm absorb considerably less than those between 200nm and 600 nm. Thus, both the absorption of the incident light and theeffect on the fluorescence of a dye are minimal when exciting above 600nm. Preferred excitation wavelengths for biological fluids, includingurine, blood, serum or plasma is 600 nm or greater. Particularlypreferred excitation wavelengths above 600 nm are those which correspondto the maximum light output of laser diodes and light emitting diodes.Preferred emission wavelengths are those above 600 nm. The intrinsicsample fluorescence can cause a high background signal if the emissionwavelength of the dye and the sample are overlapping. In addition, thescattered light of the excitation source can also contribute to thebackground signal. The contribution of the scattered light to thebackground can be seen, for example, in FIG. 5. In general, themagnitude of the scatter is inversely proportional to the fourth powerof the measured wavelength. This teaches that desired emissionwavelengths are in the near-infrared or in the infrared region of thespectrum. The inventive teachings described herein provide for dyes anddye systems which excite above 600 nm and which emit above 650 nm andmore preferred, above 730 nm.

The Stokes shift of the dye should be as large as possible to minimizethe measurement of background from the excitation source so that thesignal-to-background ratio at the limit of sensitivity is maximized. Alarge Stokes shift, however, will only maximize the efficiency of thefluorescence measurement and may not always result in an accuratefluorescence measurement. For example, table 3 shows data from severaldye systems which were excited between 420 nm and 670 nm in eitherbuffer or undiluted human serum. The fluorescence intensity of the firstdye system (from line 1, table 1), when excited at 475 nm in serum, isonly 7.6% of the intensity in buffer even though the Stokes shift is 205nm. The second dye system (from line 4, table 1), excited at 420 nm, is28% of the intensity in buffer with a 260 nm Stokes shift. The third andfourth dye systems (from line 60 and line 59, table 1), excited at 670nm and 650 nm and with 110 nm and 130 nm Stokes shifts, respectively,have fluorescence intensities which are comparable in buffer and inserum. The fifth dye system, which is a hybrid phthalocyanine derivative(from line 1, table 2), has comparable fluorescence intensities inbuffer and serum when excited at 646 nm with a Stokes shift of 114 nm.The data show that the fluorescence intensity is greatly affected whenthe excitation wavelength is within the range of the absorbance of thesample in which the measurement is made. The data also show that themagnitude of the Stokes shift does not have an influence on the accuracyof the measurement. These data are representative of other dyes and dyesystems which are excited at a wavelength where the sample absorbs. Theeffect of the decreased fluorescence emission is not a result of theemission wavelength (that is, 680 nm or 780 nm) because both serum andbuffer solution absorb minimally at 680 nm and 780 nm. One skilled inthe art can appreciate, that with the inventive teachings describedherein, the wavelengths for excitation and emission of a dye systemshould be a function more of the absorption and emission characteristicsof the sample rather than selecting only a dye system with a largeStokes shift.

The availability of dyes with Stokes shifts greater than 100 nm isgreatly limited, particularly when the excitation wavelength is greaterthan 600 nm. To further limit the usefulness of available dyes, thesolubility of the dyes in aqueous samples can be a problem because mostdyes with large Stokes shifts are water insoluble.

The problem of a dye possessing a small Stokes shift is usually overcomein the engineering of the fluorometer by the use of monochromators orexpensive optics which filter out the light from the excitation source.However, to overcome the loss in light intensity due to the filters, forexample, one requires the use of high powered light sources. These lightsources produce heat which must be dissipated in an instrument by usingheat sinks or fans. The complexity of the fluorescence measuring device,both from an optical and a mechanical perspective, is thus greatlyaffected by the inadequacies of the dye system. With the advent ofnear-patient testing in hospitals and emergency departments, instrumentswhich measure fluorescence in immunoassays will be required to beportable and uncomplicated to the technician. Thus, the future state ofthe art for the manufacture of, for example, fluorometers which areemployed for immunoassays will be required to change to simple andportable instruments. The high powered light sources and expensiveoptics currently incorporated into fluorometers will not meet therequirements for small, portable instruments. The inventive features ofthe instant invention teach that fluorescent labels can be prepared withlarge Stokes shifts and be tuned to wavelengths both of which arecompatible with excitation sources and emission detectors and which arecompatible with the absorption and emission of the sample, for example,blood, serum, plasma, urine, ground water, and the like. The excitationand emission wavelengths of the novel fluorescent particles cangenerally be varied independently of each other.

The dye must be compatible with the fluid phase of the assay, or inother words, the dye must be water soluble or water insoluble dependingon the visualization or assay format. Many fluorescent dyes are waterinsoluble or poorly water soluble and these dyes are not easily used forlabelling molecules, proteins, nucleic acids or cells. One skilled inthe art will recognize that water insoluble dyes can be incorporatedinto latex particles as described in U.S. Pat. Nos. 4,326,008, 4,609,689and 5,154,887, which are hereby incorporated by reference. Thus, waterinsoluble dyes can be made useful by incorporation into latex particlesfor visualization in a variety of assay formats.

The dye should be as bright as is necessary to achieve the desiredsensitivity. If one knows the extinction coefficient and the quantumyield of the dye and the concentration of the target to be measured, itcan be estimated whether the dye is bright enough to achieve the desiredsensitivity. Incorporation of dyes into latex particles or theutilization of an enzyme which catalyzes the production of a fluorescentsubstrate are examples of techniques which one skilled in the art usesas amplification systems.

The instrument used to detect the fluorescent signal is generallydesigned around the specifications of the dye and the specimen or samplebeing visualized or assayed because of the limited numbers of dyes whichcan be successfully used. As discussed above, the components of theinstrument are selected for a particular dye system since a usefulinstrument must be highly tuned to eliminate the light from theexcitation source.

Each of the conditions described above, taken together, greatly narrowsthe development of dye systems which can be employed for measuringsub-picomolar concentrations of analytes, particularly in biologicalfluids. The limitations also impose restrictions on the design of aninstrument to measure the fluorescence. The novel teachings of theinstant invention allow the design, synthesis and tuning of dye systemsto match, generally, nearly any instrument design.

Several inventive teachings are described for tuning excitation andemission wavelengths of dyes so that the excitation and emission arecompatible with the sample matrix in which the fluorescence is measuredand the instrument for quantifying the fluorescence. One teaching is toeither incorporate or adsorb at least two dyes into or onto particles,which, as a pair, exhibit fluorescence energy transfer. The particleswhich can be used are those which absorb dyes on the surface or insidethe particle. Another teaching is to incorporate dyes which arecovalently attached to each other and which also exhibit fluorescenceenergy transfer both in solution and in particles. Yet another teachingis to incorporate hybrids of phthalocyanines, naphthalocyanines,anthranylocyanines and derivatives of these classes of compounds.

The selection of dye pairs for incorporation into particles is based ontheir ability to exhibit energy transfer (singlet-singlet energytransfer) at the appropriate excitation wavelength of the donor dye andthe emission of the acceptor. Fluorescence energy transfer of twomolecules is well known to those skilled in the art and the rate ofenergy transfer is described by Forster in Ann. Physik. (1948) 2,55-75.Fluorescence energy transfer has been used as a spectroscopic ruler topredict proximity relationships in proteins, RNA and peptides (AnnualReview of Biochemistry (1978), 47, 819-846) and also to probegeometrical details in particles (Physical Review Letters (1988) 61,641-644). U.S. Pat. No. 5,326,692 describes fluorescent particles withcontrollable enhanced Stokes shifts. U.S. Pat. Nos. 4,542,104 and4,666,862 describe fluorescence energy transfer in phycobiliproteins.These dye complexes are described for use as labels in immunoassays;however, the limited use of phycobiliproteins and the expense of thesenatural protein complexes make them undesirable for use on a commercialscale. Unsymmetrical or hybrid phthalocyanines have been described, forexample, in J. Am. Chem. Soc. 1990, 112, 9640-9641, Chemistry Letters1992, 2031-2034 and Inorg. Chem. 1994, 33, 1735-1740 but the inventiveteachings described herein expand the potential compounds which can besynthesized for use in immunodiagnostics to achieve adequatefluorescence intensities and desired excitation and emissioncharacteristics. The inventive teachings described herein also teachthat the ratio of the various diiminoisoindiline or dicarbonitrileprecursors and their substitution by electron donating or electronwithdrawing groups in the synthesis of the hybrid phthalocyanines,naphthalocyanines and anthranylocyanines will affect the absorptionspectrum and the excitation and emission wavelengths of the compounds.

In one aspect, the novel fluorescent particles of this invention arecomposed of at least two dyes which are positioned in the interior or onthe exterior of particles at an energy exchanging distance. One skilledin the art will recognize that various particles can be utilized, suchas latex, silica, alumina, liposomes, various colloids and the like.Particularly preferred particles are latex particles. The selection ofthe dye molecules for incorporation into the particles should be relatedto the specific use of the particles, the sample to be analyzed and theinstrument for measuring the fluorescence. For example, when developingan assay for an analyte in a biological medium, such as serum or a cellextract, the intrinsic absorbance and fluorescence of the sample must beconsidered. Serum and cellular components absorb in the ultravioletspectrum as well as in the visible spectrum up to around 600 nm and theintrinsic fluorescence can broadly approach 600 nm. In addition, sampleswhich contain small particles, such as dirt particles in ground water,lipoproteins in serum or blood, cells and cellular particles andcomponents will scatter the excitation light which results in a higherbackground signal. The ideal dye couple would include the donor dyewhich would be excited or absorb at above 600 nm and emit at awavelength which the acceptor dye absorbs, and the acceptor dye shouldemit at a wavelength above 600 nm. In the case of a single dye system,for example, with the use of hybrid phthalocyanine derivatives, theexcitation and emission wavelengths should also be above 600 nm. Thesample, for example, serum, then does not affect fluorescence of theacceptor dye because the sample poorly absorbs at the absorption of thedonor dye and the acceptor dye emits at a wavelength where the sampledoes not fluoresce.

Fluorescent dye molecules incorporated into or onto particles willexhibit fluorescence quenching because of the close proximity of thedyes to each other and to the matrix of the particle. When loading dyesinto or onto particles, one must optimize the concentration of dye as itrelates to quenching. The dyes can be loaded successively or together.The degree of quenching can be quantified by measuring the fluorescenceemission of a dilute suspension of particles (about 0.001% to 0.1%solids) in a buffer solution, in a buffered protein solution or in waterand then also measuring the fluorescence of the same concentration ofparticles in solvent which liberates the dyes from the particles. Theratio of the fluorescence intensities (1- fluorescence intensity ofincorporated dyes divided by the intensity of liberated dyes! is thedegree of quenching of the dyes in the particle. In practice, oneincorporates dyes at various concentrations and measures thefluorescence intensities of the incorporated and liberated dyes tooptimize the intensity of fluorescence of the particle while minimizingthe quenching of fluorescence in the particle. In a situation where morethan one acceptor dye is used to minimize fluorescence quenching and tomaximize fluorescence intensity, one may use different acceptor dyeswhich have emission peaks which are within about 10 nanometers of oneanother. Another important consideration is the efficiency of thefluorescence energy transfer. In practice, if the energy transferefficiency is not close to 100%, then one can observe the fluorescenceof the donor dye. The resulting fluorescence of the donor dye can makethe particles undesirable or even useless because the "effective Stokesshift" (that is, the shortest wavelength distance to a light source fromthe defined acceptor molecule emission wavelength in the fluorescencesystem) of the particles is now not the difference between theexcitation and emission wavelengths of the donor and acceptor dyes,respectively, but rather the difference between the donor emission andthe acceptor emission wavelengths. The emissions of the donor andacceptor wavelengths can overlap partially with each other whenefficient energy transfer is not obtained and complicate the selectionof filters for use in a fluorometer. The decrease in the energy transferefficiency can also be directly related to a decrease in the emission ofthe acceptor dye, resulting in a particle which may not be as bright asa particle with efficient energy transfer. In addition, under conditionsof inefficient energy transfer, slight changes in the sample or insolution conditions, for example, pH, ionic strength and the like, mayaffect the magnitude of energy transfer efficiency and thereby mayaffect the intensity of the fluorescent signal.

In selecting dye pairs for fluorescence energy transfer one begins bystudying the overlap of the donor emission and acceptor excitationwavelengths. The dyes are positioned in the particle at an energyexchanging distance from one another which allows singlet-singlet energytransfer. Although a particular pair of dyes has acceptable overlappingexcitation and emission wavelengths (for example, see Proc. Natl. Acad.Sci. USA 1969, 63, 23-30), they may not exhibit fluorescence energytransfer in particles or they may have suboptimal (less than 80%)efficiency of energy transfer. The process to determine whether 2 ormore dyes will exhibit efficient energy transfer is throughexperimentation after the appropriate spectral overlap criteria are met.The efficiency of fluorescence energy transfer is determined bymeasuring the fluorescence intensity of the donor dye alone in particlesand also measuring the fluorescence emission of the particles which haveincorporated 2 or more dyes (that is, the fluorescent energy transferparticle) at the emission wavelength of the donor dye, both sets ofparticles having the same concentrations of donor dye and particles. Themeasured fluorescence at the donor dye emission wavelength of thefluorescent energy transfer particles divided by the fluorescence of thedonor dye particles is the efficiency of fluorescence energy transfer.Ideally, in practice, the emission of the donor dye should beundetectable or only slightly detectable so that the effective Stokesshift is not reduced because of the donor dye emission. Preferredfluorescence energy transfer efficiencies are 80% or greater andparticularly preferred fluorescence energy transfer efficiencies are 90%or greater.

The inventive teachings described herein provide for particles withreduced quenching and improved fluorescence intensities. A largemajority of fluorescent molecules have aromatic character, that is, theypossess 4n+2 pi electrons. The resultant aromatic character promotesstacking of the molecules, especially of water insoluble molecules inaqueous solutions or in particles in aqueous solution, which in turnpromotes fluorescence quenching. The novel fluorescent particlesdescribed in the instant invention are incorporated with dyes which,through steric interference of the dye molecules, have a minimizedpropensity to stack in the particles. In another aspect of thisinvention, fluorescence quenching of dye molecules in particles isminimized by employing different dyes with approximately the sameexcitation and emission wavelengths. That is, the wavelength maximum forexcitation and/or emission of the different dyes are within about 10 nmof each other so that there is substantial overlap of the peaks. Oneskilled in the art can appreciate that the width of excitation andemission spectra of various dyes can vary. The principle here is thatdifferent dyes will not stack in an organized orientation with eachother to the same degree as dyes which are the same. An analogy to thisstacking principle is the depression of the melting point of a purecompound by an impurity. It is well known to physical chemists that animpurity in a solid compound lowers its melting point because theimpurity disrupts the formation of the crystal lattice of the purecompound. Incorporating dyes into or onto particles using organicsolvents and then removing the solvent causes the dye to precipitate orcrystallize in the particle. The disruption of the crystalline latticeof dye molecules in particles will alter the stacking of the moleculesand thereby reduce quenching. Thus, incorporation of dissimilar dyemolecules with similar excitation and emission spectra improvesfluorescence intensities of the particles by decreasing the quenchinginteractions of the molecules.

In another aspect of this invention, incorporation into particles ofdissimilar dyes which exhibit fluorescence energy transfer in theparticles may also disrupt the other's crystalline lattice formation.Thus, the fluorescence intensities of particles exhibiting fluorescenceenergy transfer will be improved as a result of decreasing quenching inthe particle because the stacking of similar dyes in the particles isdisrupted by the dissimilar dye.

In yet another aspect of this invention, the synthesis of phthalocyaninederivatives and hybrid phthalocyanine derivatives with axial ligandsreduces the stacking of the aromatic ring system, thus minimizing theinteractions between molecules and maximizing fluorescence intensities.

One skilled in the art can appreciate that more than one dye pair whichexhibits fluorescence energy transfer can be incorporated into or ontoparticles resulting in a class of particles which fluoresce at differentwavelengths. In addition, with the inventive teachings described herein,incorporation into or onto particles of 3 or more dyes, which togetherprovide a cascade of energy transfer from the absorber to theintermediate donor to the acceptor (which fluoresces), can result in theproduction of particles with very long Stokes shifts and allows one toproduce particles with nearly an unlimited variety of excitation andemission characteristics.

FIG. 1 shows preferred acceptor dyes which are phthalocyanines,naphthalocyanines and anthranylocyanines. FIG. 2 shows particularlypreferred acceptor dyes which are derivatives of siliconphthalocyanines, naphthalocyanines and anthranylocyanines, where R ishydrogen or an alkylcarbon chain from 1-20 carbons, either saturated orunsaturated, having 0-10 heteroatoms (N,O,S), and having 0 or 1 siloxidegroups. The best mode compounds are those in which R=

Si(CH₃)₂ C₆ F5

Si(C₆ H₁₃)₃

Si(CH₃)₂ (CH₂)₃ CN

Si(CH₃)₂ (CH₂)₁₀ COOCH₃

Si(CH₃)₂ CH═CH₂

Si(CH₃)₂ (CH₂)₁₀ COOH

Si(CH₃)₂ (CH₂)₄ Cl; and

Si(CH3)₂ (CH₂)₆ CH═CH₂.

The parent compounds of the phthalocyanines and naphthalocyanines arepreferred because their emission wavelengths are around 680 nm and 780nm in latex particles, respectively. Also preferred parent compounds arethe anthranylocyanines which have emissions around 850 to 900 nm. Thesethree classes of parent compounds will collectively be called"phthalocyanine derivatives" and may or may not have an included metaland may or may not have axial ligands. The emission wavelengths for thephthalocyanine derivatives are particularly useful for quantifyingfluorescence in biological samples and for minimizing the backgroundscatter intensity. Those skilled in the art can appreciate thatphthalocyanine derivatives can be synthesized, for example, byderivatization of the phenyl, naphthyl or anthranyl rings with varioussubstitutes to yield different molecules but these variants, also arewithin the scope of the instant invention. Derivatives oftetraazaporphine are also within the scope of the instant invention. Thederivatization of the aromatic structure can produce blue or red shiftedexcitation or emission wavelengths. The choice of the donor dye toexcite the phthalocyanine derivative dyes is dependent on having a donordye emission wavelength which corresponds to the appropriate range ofabsorbance wavelengths of the phthalocyanine derivative. FIG. 3 showsthe absorbance spectra of the silicon dihydroxyphthalocyanine andsilicon dihydroxynaphthalocyanine in dimethylformamide. A potentialrange of excitation of the these acceptor dyes by the donor dye isbetween approximately 550 nm and 670 nm and 600 nm and 760 nm,respectively. One skilled in the art will recognize that many dyes wouldbe candidates for the donor dye because of the wide useful range ofwavelengths which can excite the acceptor dyes. The choice of theacceptor dye should meet the criteria outlined above. Several examplesare described which illustrate the versatility of this novel approach.Assume that an instrument is to be built with an excitation source whichhas a maximum intensity at 480 nm and a detector which has a goodquantum efficiency at 600 to 700 nm. The donor dye should thus becapable of being excited at 480 nm and further assuming that aphthalocyanine derivative is the acceptor dye for emission at 680 nm,the donor should then emit in the range of 550 to 670 nm.

Preferred classes of dyes for this application are styryl,phenylbutadienyl and phenylhexatrienyl dyes. Styryl dyes are those ofthe following formula: ##STR1## and phenylbutadienyl dyes are of theformula: ##STR2## and phenylhexatrienyl dyes are of the formula:##STR3## wherein R1, R2 and R3 can be the same or different and R1, R2and R3 are H or alkylcarbon chains from 1-20 carbons, either saturatedor unsaturated, and having 0-10 heteroatoms (N, O, S).

In general, these dye classes excite approximately between about 470 and530 nm and emit approximately between 600 and 780 nm (see MolecularProbes Handbook of Fluorescent Probes and Research Chemicals by RichardP. Haugland, 1992-1994, p. 156). A particularly preferred styryl dye isthe trans-4- 4-(dibutylamino)styryl!-1-methylpyridinium iodide (AldrichChemical Co.) which has its maximum absorbance at 486 nm indimethylformamide and its emission at 600 nm. One skilled in the artwill recognize that the substituents off the aniline nitrogen and thepyridium nitrogen of these classes of dyes can vary and that preferredsubstituents are those with hydrophobic groups to maintain waterinsolubility.

In another application of this novel technology, an instrument system isbuilt which has a source of maximum intensity at 420 nm and a detectoras described in the above example. The dye system here can include thephthalocyanine acceptor; however, a different donor must be employed. Apreferred donor for this application is ameso-tetra-2-aminophenylporphine (Porphyrin Products, Inc., Logan Utah)which has a maximum absorbance for excitation at 418 nm indimethylsulfoxide and an emission around 655 nm. This porphyrin willexcite the phthalocyanine derivative in latex particles and the dyesystem will emit at 680 nm.

In a particularly preferred application, an instrument system is builtto perform immunoassays in neat blood or serum or in various biologicalspecimens. The excitation source is an LED or laser diode which has itsmaximum intensity around 650 nm to avoid absorption of the light by theblood or serum sample. The detector has good quantum efficiency at 700to 800 nm so a preferred acceptor dye is a naphthalocyanine derivativewhich has an emission at approximately 780 nm, an emission wavelengthwhich is generally not in common with blood or serum samples orbiological specimens. A donor dye for the naphthalocyanine acceptorshould absorb at around 650 nm to coincide with the source and emitbetween approximately 660 nm and 760 nm. Preferred classes of dyes forthis donor application are the carbocyanine dyes and theethenyl-substituted dipyrrometheneboron difluoro dyes, as described inU.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.

In yet another particularly preferred application, for immunoassays inneat blood or serum, the excitation source is around 790 nm and theemission wavelength is around 900 nm. A preferred dye for a single dyesystem is a silicon 1,6-octaethoxynaphthalocyaninebis(dimethylhexylvinylsilyloxide) which is excited at 790 nm and emitsat about 900 nm.

Preferred dyes for use as donor dyes for naphthalocyanines andnaphthalocyanine derivatives are, carbocyanines and ethenyl-substituteddipyrrometheneboron difluoro dyes, as described in U.S. Pat. Nos.5,187,288, 5,248,782 and 5,274,113 which have excitation wavelengths upto 790 nm and emission wavelengths between about 670 nm and 800 nm.

Preferred carbocyanine dyes, which generally excite between 500 and 750nm (see Molecular Probes Handbook) are of the general formula: ##STR4##wherein n is 1 or 2; or 3; wherein R1 and R2 are S, N, or O; and whereinR3 and R4 are H or alkylcarbon chains of from 1-20 carbons, eithersaturated or unsaturated and having 0-10 heteroatoms (N, O, S).

Also preferred carbocyanine dyes are also of the general formula:##STR5## wherein n is 1 or 2; or 3; wherein R1-R6 are H or alkylcarbonchains of from 1-20 carbons, either saturated or unsaturated and having0-10 heteroatoms (N, O, S).

Preferred donor dyes are also the ethenyl-substituteddipyrrometheneboron difluoro dyes, which generally excite above 500 nm(see Molecular Probes Handbook) and are of the general formula asdepicted in FIG. 4, wherein R1-R7 include substituents as described inU.S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.

Particularly preferred donor dyes are1,1'-dihexyl-3,3,3',3'-tetramethylindocarbocyanine iodide,1,1'-diethyl-3,3,3',3'-tetramethylindodicarbocyanine iodide and(E,E)-3,5-bis-(4-phenyl-1,3-butadienyl)-4,4-difluoro-4-bora-3a,4a-diazo-5-indacene (from Molecular Probes Inc., Eugene, Oreg.) whichhave absorption maximums of 642 nm, and 645 nm and 650 nm and emissionmaximums of 674 nm and 665 nm, and 670 nm, respectively, indimethylformamide. Particles incorporated with these particularlypreferred dyes and a naphthalocyanine derivative will excite with a 650nm source and emit at approximately between 780 nm and 870 nm. Oneskilled in the art will recognize that the excitation and emissionspectra for any particular dye has a Gaussian form and therefore theexcitation source does not need to correspond exactly to the excitationmaximum of the donor dye in order to obtain an intense fluorescentsignal. Likewise, the donor emission does not have to coincide with thehighest absorption of the acceptor dye in order to achieve efficientenergy transfer. One skilled in the art will also recognize that thesubstituents at and on the 1 and 3 positions of the carbocyanines andthe substituents at the R1 and R7 positions of the dipyrrometheneborondifluoro dyes, and the conjugation between the ring structures can varyand these variations are also useful in tuning fluorescence spectra ofthe particles.

Also preferred emission wavelengths of fluorescent particles range fromabout 800 nm to 1000 nm. This near infra-red region is important becausethe scattering component of the light decreases substantially, thuslowering the background of the fluorescent measurement. In addition,biological samples do not absorb or fluoresce substantially in the 800nm-1000 nm range. Particulate materials in the samples, for example,lipoproteins in serum, particles in ground water, cellular debris inbiological samples and the like, can increase the background signalbecause of scattered light and the measurement of the scattered light isminimized in the 800-1000 nm range. For example, FIG. 5 illustrates theattenuation of the background signal as the wavelength of the measuredlight increases from 730 nm to 900 nm in an immunoassay device, asdescribed in allowed application Ser. No. 07/887,526 now U.S. Pat. No.5,458,352 (which is herein incorporated by reference), containing eitherneat human serum or no serum. This figure shows that the backgroundsignal decreases by a factor of 5 when measuring at 900 nm as comparedto 790 nm when the illumination source is a 1 milli watt ("mW") 670 nmlaser diode. In addition, excitation of neat serum at 670 nm does notresult in a significant measurable fluorescence between 730 nm and 900nm. Thus, for example, the signal to background ratio of the measurementof fluorescence of a dye which emits at around 900 nm as compared to adye emitting at around 790 nm would be improved by a factor of 5. Thesignal to background ratio improves by a factor of about 30 whenmeasuring emission at 780 nm as compared to 730 nm (see FIG. 5).Maximizing the signal to background ratio, in general, is commonlysought in analytical chemistry because the sensitivity of themeasurement is improved. Preferred dyes, for example as described in J.Chem. Soc. Perkin Trans. 1, (1988), 2453-2458, which emit above 780 nminclude derivatives of the naphthalocyanine and anthranylocyanineclasses (FIG. 1) and the naphthalocyanine class is characterized by thegeneral formulae, as depicted in FIG. 6, where M is a metal such as Si,Ge, Al, Sn and Ti and the like, and where R is an axial ligand, alkyl oraryl with or without a silicon (preferred axial moieties are synthesizedfrom alkyl or aryl silyl chlorides), and where X is an electron donatinggroup or groups which can be the same or different, including, such asamino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like. Theelectron donating character of the X group or groups red-shifts theemission wavelength as compared to the general naphthalocyaninecompounds (FIG. 1). For example, the compounds described in examples 26,27 and 28 are illustrative of dyes which have emission wavelengthsaround 850 nm. These preferred dyes would yield an improved signal tobackground ratio as compared to dyes emitting at 780 nm (See FIG. 5).Electron withdrawing groups can also be utilized for the X groups, suchas halogen, nitro, cyano, sulfate, carboxyl and carboxyalkyl and thelike, which will blue shift the excitation or emission wavelengths.Preferred donor dyes for this class of near infra-red emitting dyes arethose which have emission wavelengths which correlate to the absorbancecharacteristics of the acceptor dye. Preferred dyes for this applicationare the ethenyl-substituted dipyrrometheneboron difluoride dyes, asdescribed in U. S. Pat. Nos. 5,187,288, 5,248,782 and 5,274,113.

Preferred molar ratios of donor to acceptor dyes in the latex particlesgenerally range from about 20:1 to about 1:20 and particularly fromabout 1:1 to 6:1. The desired fluorescence intensity should be obtainedthrough experimentation by incorporating various ratios of donor toacceptor dyes into the particles at various dye concentrations andmeasuring the fluorescence emission of the particles.

The geometrical orientation of the donor and acceptor dyes will affectthe efficiency of energy transfer between them. The donor and acceptordyes can be synthesized to form a compound of optimal geometry, which,in solution, exhibits efficient fluorescence energy transfer ("FET").The optimized FET compound then may be incorporated into particles.Phthalocyanine derivatives can be utilized for this application for theacceptor moiety, where the phthalocyanine derivative can be substitutedwith electron donating or withdrawing groups (as described above) toaccomodate the desired excitation and emission wavelength. For example,preferred naphthalocyanine compounds for this application are those asdepicted in FIG. 7, where X is hydrogen or electron donating groups,such as amino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl and the like andD is the donor dye covalently attached to the naphthalocyaninederivative at a distance which allows for energy transfer between thedonor and acceptor. With the inventive teachings of the instantinvention, one skilled in the art will recognize that all phthalocyaninederivatives can function as donor or acceptor molecules. For example, asilicon ortho octaethoxy(phthalocyanine) derivative will emit atapproximately 750 nm to 780 nm, similar to a silicon naphthalocyaninederivative. Generally, the distances between donor and acceptor areabout 5 angstroms to 60 angstroms, and preferably from 5 angstroms to 15angstroms. In addition, each naphthalocyanine derivative can have 1-4donor dyes attached, depending on the required application of the FETcompound. Suitable donor dyes are those which emit in the absorbancerange of the acceptor dye. Example 29 describes the synthesis of afluorescein-silicon phthalocyanine FET compound. Table 1, item 56, showsthe fluorescence characteristics of this compound in latex particles.One skilled in the art will appreciate that with the inventive teachingsdescribed herein, many FET compounds may be synthesized for manyparticular applications requiring specific excitation and emissionwavelengths.

Another approach to developing particles which exhibit desired andpredictable fluorescence properties in the high visible to near infraredspectrum is to synthesize unsymmetrical or hybrid phthalocyanines,naphthalocyanines or anthranylocyanines and their derivatives. The term"hybrid phthalocyanine derivatives" will herein refer to all classes ofhybrid phthalocyanines, naphthalocyanines and anthranylocyanines andtheir derivatives, with or without metal and axial ligands, includingtetraazaporphines and their derivatives. The novel hybrid moleculesdescribed herein appear to exhibit intramolecular energy transfer. Thehybrid phthalocyanine derivatives can be synthesized fromdiiminoisoindoline or derivatives of diiminoisoindolines and incorporatea metal, for example, silicon, and elaboration with axial ligands orthey can be synthesized from dicarbonitrile derivatives of benzene,naphthalene or anthracene compounds, respectively, for subsequentinclusion of various metals and elaboration with axial ligands. Hybridmolecules also comprised of derivatives of tetraazaporphines, asdescribed in Inorg. Chem. (1994), 33, 1735-1740, are also within thescope of the hybrid phthalocyanine derivatives of the instant invention.A synthetic strategy for hybrid phthalocyanine derivatives with 2different subunits is described, for example, in J. Am. Chem. Soc.(1990), 112, 9640-9641, Inorg. Chem. (1994),33, 1735-1740, Chem.Letters, (1992), 763-766, Chem. Letters, (1992), 1567-1570 and Chem.Letters, (1992), 2031-2034. These papers refer to the synthesis ofhybrid molecules with zinc metal or without metal and without axialligands. The versatility of the synthetic approach described herein asit applies to the inventive teachings of the instant invention is thatthe character of the diiminoisoindoline and its derivatives will dictatethe excitation and emission characteristics of the molecule and,furthermore, elaboration with axial ligands will minimize quenching bydecreasing stacking in the particles and maximize fluorescenceintensity. Axial ligands on hybrid phthalocyanine derivatives are alsobeneficial on water soluble compounds because the axial ligands willminimize interaction of the hybrid molecule with, for example, proteins,antibodies and nucleic acids, which may or may not be covalently coupledto the hybrid molecule.

Novel hybrid phthalocyanine derivatives are described herein, whichcontain 3 or 4 different subunits, and allow for larger Stokes shifts.The principle is that excitation occurs with the subunit which has thehighest energy or the lowest wavelength absorption and the emissionoccurs in the lowest energy subunit.

The desired excitation and emission wavelengths of the hybridphthalocyanine derivative will determine the types of diiminoisoindolinederivative and dicarbonitrile derivative precursors which are used inthe synthesis of the hybrid phthalocyanines. The desired excitation andemission wavelengths are generally dictated by the sample, the type offluorescent measurement and the instrument. Various combinations ofdiiminoisoindoline derivative and dicarbonitrile derivative precursorsmay also combine to form a hybrid phthalocyanine derivative which mayhave a red shifted or blue shifted excitation and/or emission wavelengthpattern. In general, electron donating substituents on thediiminoisoindoline or dicarbonitrile precursors, particularly situatedat the ortho positions (that is, ortho to the tetraazaporphine structureas indicated in FIG. 6 for the X substituents) of the phthalocyaninestructure, such as amino, hydroxyl, alkoxy, aryloxy, phenyl, alkyl andthe like, will red shift the excitation and/or emission wavelengths.Conversely, generally, electron withdrawing substituents, alsoparticularly at the ortho positions, such as halogen, nitro, cyano,sulfate, carboxyl and carboxyalkyl and the like, will blue shift theexcitation or emission wavelengths. In addition, positions on thesubunits other than the ortho positions can affect the excitation andemission characteristics of the hybrid phthalocyanine derivative. Thechoice of either diiminoisoindoline or dicarbonitrile precursors for thesynthesis of the hybrid phthalocyanine derivatives is generally relatedto the desired presence or absence of metal and the type of metal in thehybrid molecule. For example, when using the diiminoisoindolineprecursors in the synthesis, a silicon metal can be incorporated duringthe tetramerization reaction to form the phthalocyanine derivativestructure. The silicon can be further modified to a silicon dihydroxyphthalocyanine derivative molecule so that axial ligands can beelaborated with, for example, various silyl chloride reagents. Theimportance of axial ligands in reducing quenching and maximizingfluorescence intensity is evident for bothphthalocyanine/naphthalocyanine molecules and the hybrid phthalocyaninederivatives (see example 31). The axial ligands can also be useful forfurther elaboration of the molecules, for example, for attaching anotherfluorescent molecule, for attaching to a protein, polypeptide or nucleicacid or for changing the charge of the molecule using sulfate,carboxylic acid or amino substituents which can affect solubility of themolecule. When using the dicarbonitrile precursors, the phthalocyaninederivative is synthesized without metal, but various metals cansubsequently be included, for example, Ge, Al, Sn, Ti and the like.These metals can also be elaborated with axial ligands, depending on thevalence of the metal.

The fluorescence quenching character of the hybrid phthalocyaninederivatives are particularly preferred over the phthalocyaninederivatives. Example 32 is a typical example of comparison of thequenching characteristics in latex particles of silicon2,3-naphthalocyanine bis(dimethylhexylvinylsilyloxide) and silicondi(1,6-diphenylnaphthalocyanine)! diphthalocyanine bis(dimethylhexylvinylsilyloxide). The hybrid phthalocyanine derivative hasessentially no quenching as compared to up to 50% quenching of thenaphthalocyanine derivative for the various dye loading concentrationslisted in the table. The fluorescence intensities of latex containingthe hybrid phthalocyanine derivative are much greater than thephthalocyanine derivative. This illustrates the special properties ofthe hybrid phthalocyanine derivatives.

The tetramerization reactions of the diiminoisoindoline ordicarbonitrile precursors to form the hybrid phthalocyanine derivativescan be directed so that opposing subunits can be the same. This isaccomplished, for example, with the use of bulky substituents on theprecursors so that in the tetramerization reaction, like subunits withbulky substituents cannot be adjacent because of steric considerations.Bulky phenyl substituents have been used on dicarbonitrile precursors todirect the precursors tetramerization to be opposing subunits asdescribed in Inorg. Chem. (1994),33, 1735-1740, Chemistry Letters(1992), 2031-2034 and Chemistry Letters (1992),1567-1570. Thesereferences, however, do not describe the synthesis of the novelphthalocyanine derivatives described herein using diiminoisoindolineprecursors with or without axial ligands.

Preferred hybrid phthalocyanine derivatives have similar opposingsubunits so that two different subunits comprise the structure.Particularly preferred hybrid phthalocyanine derivatives have similaropposing subunits on one axis and different opposing subunits on theother axis. The nature of the particularly preferred molecules is thatred or blue shifted excitation or emission wavelengths and a longerStokes shift can result because of the selection of the precursormolecules for the tetramerization reaction. For particularly preferredhybrid phthalocyanine derivatives, for example, the "donor"diphenyldiiminoisoindoline or the diiminoisoindoline precursors wouldcontribute to 650 nm absorbance of the hybrid molecule, and thereby tothe excitation of the hybrid molecule. The diphenylphenyldiiminoisoindoline or the phenyldiiminoisoindoline precursorswould act as an "electron transfer subunit" to the "acceptor subunit",which would be a dialkoxy or aryloxy phenyldiiminoisoindolineprecursors, so that emission is dictated at the lowest energy by theacceptor subunit at about 850 nm. The nature of the "electron transfersubunit" is important because it is not desirable for this subunit toemit because then the desired emission of the acceptor subunit will nottake place. Thus, the HOMO and LUMO character of the electron transfersubunit should be designed with reference to the donor and acceptorsubunit molecules. The relationship of the energies of the HOMO and LUMOas they relate to excitation and emission are taught by Pariser et al,J. Chem. Phys. (1953), 21, 767-776, by Pople, Trans. Faraday Soc.(1953), 49, 1375-1385, by McHugh et al, Theoret. Chim. Acta (Berlin)(1972), 24, 346-370 and by Kobayashi et al, Inorg. Chem. (1994),33,1735-1740, Chemistry Letters (1992), 2031-2041, Konami et al, MolecularPhysics (1993), 80, 153-160.

Another application requires the hybrid molecule to have two excitationwavelengths, one at approximately 650 nm and another at about 680 nmwith emission for both excitations at about 760 nm. Thus, the precursorsresponsible for the excitation could be a diiminoisoindoline for the 650nm and a tetrafluorodiiminoisoindoline for the 680 nm excitations. Theemitting subunit, which can also be used to direct the tetramerizationreaction so that the emitting subunits are opposed in the molecule, canbe a diphenyl phenyldiiminoisoindoline. The excitation and emissionwavelengths of the resulting hybrid phthalocyanine derivative are thusgenerally representitive of the individual diiminoisoindolineprecursors.

Yet another application requires excitation at about 650 nm and emissionat about 750 nm. The precursors responsible for excitation and emissioncould be diiminoisoindoline and diphenyl phenyldiiminoisoindoline,respectively. The latter precursor also acts to direct the emittingsubunits to be opposed.

In another application, a large extinction coefficient at the excitationwavelength is desired for excitation at about 650 nm. The emissionwavelength should be at about 850 nm. The precursors responsible forexcitation could be a diphenyldiiminoisoindoline, which would directthese subunits to be opposed and thereby two subunits would contributeto provide the desired extinction coefficient. Aphenyldiiminoisoindoline derivative precursor could act as an electrontransfer subunit and an alkoxy phenyldiiminoisoindoline precursor couldbe the acceptor with a characteristic emission at about 850 nm.

In another application, two emission wavelengths are desired from acompound which is excited at a single wavelength. The desired excitationis around 650 nm and the emission should be around 760 nm and 810 nm.The precursor responsible for excitation could be atetrafluorodiiminoisoindoline or atetrafluorobenzene-1,2-dicarbonitrile. The precursor responsible foremission could be a dibutoxyphenyldiiminoisoindoline or a 3,4-dibutoxynaphthalene-1,2-dicarbonitrile, respectively.

The resulting compounds are then incorporated into particles to yieldparticles which exhibit excitation wavelengths above about 600 nm andemission wavelengths above about 650 nm. One skilled in the art willalso appreciate that water soluble hybrid phthalocyanine derivatives arevaluable for coupling to proteins, polypeptides, nucleosides, nucleicacids and the like, for detecting their presence in biological fluids orfor performing DNA probe or immunoassays.

Preferred particle sizes range from about 0.1 nm to 5000 nm andpreferably from about 1 nm to 1000 nm. The choice of particle sizeshould be related to the specific function for the label. The particlesize may vary for a particular application. For example, in animmunoassay, if the label requires a more intense fluorescence formeasuring very low concentrations of analytes, then one would employlarger particles because larger particles can incorporate more dyemolecules. The small particle sizes (0.1-1 nm) may be employed influorescence polarization assays, as described for example, in U.S. Pat.Nos. 4,420,568, 4,476229 and 4,510,251, in in vitro visualization ofcellular components or in in vivo imaging techniques.

The resulting fluorescent dye particles which exhibit the appropriateexcitation and emission characteristics are further adsorbed orchemically reacted with various nucleic acids, nucleotides, proteins orpeptides and the like which are required for a specific purpose. Theadsorption of macromolecules to particles, particularly latex particlesis well known to those skilled in the art and generally involvesadsorption of the macromolecule at a temperature between 5° C. and 50°C. and at a pH which is below the pI of the molecule. For example,fluorescent particles exhibiting fluorescence energy transfer can beadsorbed with either antibodies for use in non-competitive immunoassaysor ligand analogues for use in competitive immunoassays in reactionmixtures of the assays. In the case of non-competitive assays, thereaction mixture would include at least one target ligand and at leastone class of fluorescent particles having bound thereto at least onereceptor specific for target ligand, forming an antibody (fluorescent)conjugate. In the case of competitive assays, the reaction mixture willinclude at least one target ligand, at least one receptor specific tothe target ligand, and at least one class of fluorescent particles,having bound thereto at least one ligand analogue, forming a ligandanalogue (fluorescent) conjugate. The antibody conjugates bound totarget ligands in the non-competitive reaction mixture and the ligandanalogue conjugates not bound by receptors specific to the targetligands in the competitive reaction mixture can be bound to a solidphase consisting of receptors specific to another epitope of the targetligand of the target ligand-antibody conjugate complexes and ofreceptors specific to ligand analogues of the ligand analogueconjugates, respectively. The fluorescent conjugates unbound by thesolid phase are removed and the fluorescence of the bound conjugates ismeasured. The measured fluorescence is related to the target ligandconcentration. The various reagents described above can also be attachedcovalently to the latex particles. For example, antibodies or ligandanalogues can be attached through amine or carboxylic acids tocarboxylic acids or amines on the surface of the particles,respectively, to form stable amide linkages.

In the case of quantifying nucleic acids in samples, the novel compoundsdescribed in the instant invention are useful because of theirbrightness and because of the near infrared emission characteristics. Ingeneral, in designing an assay for a nucleic acid, one selects a probemolecule which is complementary to the nucleic acid to be quantified.The probe molecule is then labeled, generally covalently, with a signalgenerator. The signal generator can be a water soluble phthalocyaninederivative or hybrid phthalocyanine derivative or a particle with theappropriate dye system, which may exhibit fluorescence energy transferor hybrid phthalocyanine derivatives or combinations of these compounds.The labelled probe molecule is then introduced into a biological samplesuspected of containing the target nucleic acid, and the labelled probesequence assembles with the target nucleic acid. The labelledprobe/target nucleic acid can then be immobilized onto a surface whichhas immobilized another nucleic acid which is also complementary to thetarget nucleic acid. Conversely, the biological sample can be introducedto a surface which has immobilized a complementary nucleic acid forimmobilization of the target nucleic acid. The labelled probe can thenbe introduced to the system for binding to the immobilized targetmolecule. The excess labelled probe is then washed away and theresultant fluorescent intensity is correlated with fluorescenceintensity from a standard curve to arrive at a concentration of thenucleic acid in the sample.

Those skilled in the art will recognize that many variations ofimmunoassays and nucleic acid assays can be performed and the inventiveteachings in the instant invention for the use of novel dye systems canbe used to develop novel adaptations to existing technologies.

Those skilled in the art will appreciate that the novel fluorescentparticles described herein have many uses in immunoassays, fluorescencemicroscopy, in vivo imaging, in vitro cancer therapy, nucleic acidassays, cell sorters and the like.

Experimental Section

Fluorescence measurements were performed on a Perkin-Elmer model LS 50BLuminescence Spectrometer for dyes emitting up to around 780 nm. In someinstances, dyes emitting above 800 nm were measured according to Example18. The fluorescence intensities are not corrected. Absorbancemeasurements were performed on a Hewlett Packard 8452A Diode ArraySpectrophotometer.

EXAMPLE 1 Synthesis of Silicon Phthalocyanine Dihydroxide SiPc(OH)₂

A suspension of silicon phthalocyanine dichloride (1.83 g, 3.0 mmol) inpyridine (50 mL) and water (50 mL) was refluxed with stirring on an oilbath at 120° C. for 18 hours. After cooling the dark blue solid productwas filtered and the residue was washed with water (10 mL), acetone (5mL) and then dried under vacuum to afford 1.71 g of the title compound.

EXAMPLE 2 Synthesis of Silicon Phthalocyanine bis(trihexylsilyloxide)(hereinafter sometimes referred to as PcSi trihexyl)

A suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol) inanhydrous pyridine (11 mL) containing chlorotrihexylsilane (733 μL, 2.0mmol) was refluxed on an oil bath at 130° C. for 5 hours. The resultingpurple solution was allowed to cool and was evaporated. The resultingslurry was treated with ice-cold hexane (2 mL) and the dark blue solidproduct was filtered, washed with ice-cold hexane (2 mL) and was driedunder vacuum to yield 249 mg of crude product. The crude product inchloroform was purified on an Alumina column (Activity 1) equilibratedin hexane and the product was eluted with hexane/toluene (2/1, v/v) as abright blue band. The solvent containing the product was evaporated toyield 69 mg of the title compound with a mp 171° C. (lit mp 175° C.).

EXAMPLE 3 Synthesis of Silicon Phthalocyanine bis (10-carbomethoxydecyl)dimethylsilyloxide! (Hereinafter sometimes referred to as PcSi methylester)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 mL) was added(10-carbomethoxydecyl)dimethylchlorosilane (586 mg, 2 mmol) and themixture was refluxed with stirring on an oil bath at 130° C. for 5hours. The dark blue solution was allowed to cool and the solvent wasevaporated. The residue was purified on a Silica gel 60 Å columnequilibrated in hexane and the product eluted slowly as a blue band withtoluene. The toluene fraction containing product was evaporated, hexane(10 mL) was added to the residue and the blue product was filtered,washed with hexane and dried to afford 105 mg of the title compound.

EXAMPLE 4 Synthesis of Silicon Phthalocyaninebis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to as PcSivinyl)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 mL) was added chlorodimethylvinylsilane (276μL, 2.0 mmol) and the mixture was refluxed with stirring on an oil bathat 130° C. for 5 hours. The dark solution was allowed to cool and wasevaporated. The residue was purified on a Silica gel 60 Å columnequilibrated in hexane and the product was eluted with toluene as a blueband. The eluate containing product was evaporated, the residue treatedwith hexane and the dark blue solid product was filtered, washed withhexane and was dried under vacuum to afford 7.5 mg of the titlecompound.

EXAMPLE 5 Synthesis of Silicon Phthalocyanine bis (3-cyanopropyl)dimethylsilyloxide! (Hereinafter sometimes referred to as PcSi cyano)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 mL) was addedchloro(3-cyanopropyl)-dimethylsilane (328 μL, 2.0 mmol) and the mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Thepurple solution was allowed to cool and was evaporated. The residue waspurified on a Silica gel 60 Å column equilibrated in hexane. The columnwas washed with toluene and the product was eluted withtoluene/isopropyl alcohol (90/10, v/v) as a bright blue band. The eluatecontaining product was evaporated under vacuum to afford 101 mg of thetitle compound with a mp>260° C.

EXAMPLE 6 Synthesis of Silicon Phthalocyaninebis(dimethylpentafluoro-phenylsilyloxide) (Hereinafter sometimesreferred to as PcSi pentafluoro)

To a suspension of silicon phthalocyanine dihydroxide (115 mg, 0.2 mmol)in anhydrous pyridine (11 mL) was addedchlorodimethylpentafluorophenylsilane (376 μL, 2.0 mmol) and the mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Thedark green solution was allowed to cool and was evaporated. The residuewas purified on a Silica gel 60 Å column equilibrated in hexane. Theproduct was eluted with toluene as a dark blue band. The eluatecontaining the product was evaporated, the residue was treated withhexane (10 mL) and the dark blue solid product was filtered, washed withhexane and was dried under vacuum to afford 73 mg of the title compound.

EXAMPLE 7 Synthesis of Silicon 2,3-Naphthalocyanine Dihydroxide(Hereinafter sometimes referred to as NaPcSi hydroxide)

A suspension of silicon 2,3-naphthalocyanine dichloride (280 mg, 0.34mmol) in pyridine (10 mL) and water (10 mL) was refluxed with stirringon an oil bath at 130° C. for 24 hours. After cooling to roomtemperature, the dark green solid product was filtered and, the residuewas washed, successively, with water (5 mL) and acetone (2 mL). Theproduct was dried under vacuum to afford 217 mg of the title compound.

EXAMPLE 8 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylvinylsilyloxide) (Hereinafter sometimes referred to asNaPcSi vinyl)

To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11mmol) in anhydrous dimethylformamide (1 mL) was addedchlorodimethylvinylsilane (0.042 mL, 0.3 mmol), followed by imidazole(14 mg, 0.2 mmol). The mixture was stirred under argon at roomtemperature for 24 hours. The solvent was evaporated and the residue waspurified on a Silica gel 60 Å column which was equilibrated in hexane.The product was eluted with toluene as a green band. The toluenefraction containing the product was evaporated and the residue wastreated with hexane. The dark green solid was filtered, washed withhexane and was dried under vacuum to afford 26 mg of the title compound.

EXAMPLE 9 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylpentafluorophenylsilyloxide (Hereinafter sometimes referredto as NaPcSi pentafluoro)

To a suspension of silicon 2,3-naphthalocyanine dihydroxide (87 mg, 0.11mmol) in anhydrous pyridine (5 ml) was addedchlorodimethylpentafluorophenylsilane (0.188 ml, 1 mmol). The mixturewas refluxed with stirring on an oil bath at 130° C. for 5 hours. Aftercooling, the solvent was evaporated and the residue was purified on aSilica gel 60 Å column which was equilibrated in hexane. The product waseluted with toluene as a green band. The toluene fraction containing theproduct was evaporated and the residue was treated with hexane. The darkgreen solid was filtered, washed with hexane and was dried under vacuumto afford 23 mg of the title compound.

EXAMPLE 10 General Procedures for the Preparation of Dye-loaded LatexParticles of Varying Sizes

The various dyes were loaded into latex particles of varying sizesaccording to the general procedures outlined below. Two procedures aredescribed and involve swelling latex particles with aqueous solutions ofeither tetrahydrofuran or dimethylformamide prior to addition of the dyesolutions. Latex particle sizes used range from 67 nm to 783 nm and oneskilled in the art recognizes that smaller and larger particles can beused. The choice of the organic solvent used to swell the particlesdepends solely on the solubility of the various dyes in either solvent.Tables 1 and 2 of Example 15 below show the aqueous organic solventsystem and the optimum dye concentration which were used for the loadinginto particles for each dye pair or for hybrid phthalocyaninederivatives, respectively, of a selected number of dyes. One skilled inthe art recognizes that many changes can be made to these procedures toprepare particles with different degrees of fluorescence intensities andquenching by loading higher or lower amounts of dye in the particles andalso by changing the ratios of each dye pair to the other. One skilledin the art also recognizes that similar techniques are useful forincorporation of dyes into latex particles, for example, as described inU.S. Pat. Nos. 4,199,363 and 4,368,258.

Surfactant-free polystyrene sulfate latex particles in sizes rangingfrom 67 nm to 783 nm and carboxyl-modified latex ("CML") particlesranging from 200 nm to 400 nm particles were obtained throughInterfacial Dynamics Corp. Inc., Portland Oreg.

Method 1 Utilizing Tetrahydrofuran

Tetrahydrofuran (0.36 mL) was added, dropwise over a 5 minutes period,to a stirring solution of 1.6 mL of 2.5% solids of latex particles atroom temperature. The latex suspension was stirred at room temperaturefor an additional 30 minutes to swell the latex. The dye solution (0.04mL), which consists of one or more dyes at an appropriate concentrationin tetrahydrofuran, was added dropwise over 5 minutes to the stirredlatex solution, to give the loading dye concentration (in 2 mL volume)as indicated in Table 1. The latex-dye solution was stirred at roomtemperature for 30 minutes in the dark. The latex solution was thentransferred to dialysis tubing (Spectra-por, 12-14,000 molecular weightcutoff, Spectrum, Houston, Tex.) and the dye-latex solutions weredialyzed against water for 12 to 15 hours at 4° C. The dye-latexsolutions were removed from dialysis and the % solids of the solutionswas calculated from the final volume after dialysis and the startingsolids concentration.

Method 2 Utilizing Dimethylformamide

Dimethylformamide (1.33 mL) was added, dropwise over a 5 minute period,to a stirring solution of 0.6 mL of 6.7% solids of latex particles atroom temperature. The latex suspension was stirred at room temperaturefor an additional 30 minutes to swell the latex. The dye solution (0.07mL), which consists of one or more dyes at an appropriate concentrationin dimethylformamide, was added dropwise over 5 minutes to the stirredlatex solution, to give the loading dye concentration (in 2 mL volume)as indicated in Table 1. The latex-dye solution was stirred at roomtemperature for 30 minutes in the dark. The latex solution was thentransferred to dialysis tubing (Spectra-por, 12-14,000 molecular weightcutoff, Spectrum, Houston Tex.) and the dye-latex solutions weredialyzed against water for 12 to 15 hours at 4° C. The dye-latexsolutions were removed from dialysis and the % solids of the solutionswas calculated from the final volume after dialysis and the startingsolids concentration.

EXAMPLE 11 Effect of Varying Dye Loading Concentration on FluorescenceIntensity and Optimization of Fluorescence Intensity Latex Particles

The incorporation of dye into latex particles must be optimized in orderto achieve the maximum fluorescence intensity and to minimize the degreeof fluorescence quenching of the dye molecules. Fluorescence quenchingcan be significant because of the close proximity of the dye moleculesin the particles. The PcSi vinyl was incorporated into 67 nm latexparticles (polystyrene sulfate from Interfacial Dynamics Corp. (IDC),Inc., Portland, Oreg.) using method 1 (example 10) at variousconcentrations as indicated in the table below. The dye latex particleswere diluted to 0.0019% solids in either water or tetrahydrofuran foreach dye concentration. The solutions were excited at 350 nm and theemission at 680 nm was measured. The percent quenching in the particlesis: (1- fluorescence intensity in water divided by the intensity in theorganic solvent!)×100. The table below shows the fluorescenceintensities as a function of dye loading concentrations and quenchingfor each condition.

    ______________________________________                                        Loading Dye Concentration                                                     (mg/mL)        Intensity (680 nm)                                                                          Quenching (%)                                    ______________________________________                                        0.01           420           41                                               0.025          489           65                                               0.05           492           73                                               0.075          401           76                                               0.1            338           83                                               0.15           197           87                                               0.3            91            90                                               0.9            34            96                                               ______________________________________                                    

These results show that an optimum loading dye concentration gives thehighest fluorescence intensities and the lowest quenching. In this case,a dye concentration of between 0.025 and 0.05 mg/mL in the loadingsolution gives the best intensity and the least quenching. Less dye than0.025 mg/mL gives less intensity and less quenching because the spacingof the dyes begins to significantly increase and more dye than 0.05mg/mL gives less intensity and more quenching because of the increasedcloseness of the dyes in the particles. This type of experimentillustrates the procedure for optimization of fluorescence intensity andfor minimizing quenching.

EXAMPLE 12 Verification of Fluorescence Energy Transfer in LatexParticles

The latex particles which were incorporated with various dyes for energytransfer were diluted to 0.06% to 0.001% solids in water and eithertetrahydrofuran or dimethylformamide and the solutions of equal solidsconcentrations were excited at wavelengths which corresponded to theapproximate excitation maximum of the donor dye. The particles werediluted into organic solvents in order to liberate the dyes from thelatex, and therefore, disrupt any energy transfer process between thedyes in the particles. The fluorescence of the solutions in water andorganic solvent at the emission maximum of the acceptor dye or dyes wererecorded and compared. Fluorescence energy transfer was defined assignificant when the emission intensity of the acceptor was at least5-fold higher in water than in the organic solvent.

EXAMPLE 13 Effect of Varying Donor Dye Concentration With Respect toAcceptor Dye Concentration in Latex Particles on the FluorescenceIntensity of the Particles

Meso-tetra-2-dimethylaminophenyl porphyrin was made as follows. To astirring solution of meso-tetra-2-aminophenyl porphyrin (100 mg, 0.15mmol) and 37% aqueous formaldehyde (500 μL, 6.0 mmol) in tetrahydrofuran(2.5 mL was added sodium cyanoborohydride (114 mg, 1.8 mmol). Themixture was then treated with a glacial acetic acid (60 μL) over 10minutes and stirred at room temperature for 3 hours. More glacial aceticacid (60 μL) was added and the mixture stirred a further 1 hour at roomtemperature. The mixture was evaporated and the residue was purified ona Silica gel 60 Å column which was equilibrated in toluene. The productwas eluted with toluene/1% isopropanol as a dark brown band. Thefraction containing the product was evaporated and the ink-blue solidresidue dried under vacuum to afford 85 mg of the title compound.

Meso-tetra-2-dimethylaminophenyl porphyrin (Tdap synthesized from themeso-tetra-2-aminophenyl porphyrin which was obtained through PorphyrinProducts, Inc. Logan, Utah) and PcSi vinyl (example 4) were incorporatedinto 67 nm latex particles (polystyrene sulfate latex from InterfacialDynamics Inc., Portland, Oreg.) using the tetrahydrofuran method 1 ofexample 10. The molar ratio of the Tdap to the PcSi vinyl varied from1/1 to 2/1 to 6/1 in the latex loading solutions while maintaining aconstant mass (0.1 mg/mL) of PcSi vinyl in each solution. The dialyzedparticles were diluted to 0.0019% solids in water and the fluorescenceintensity at 680 nm of the PcSi vinyl was measured as a function ofexcitation wavelength between 350 nm and 470 nm. The excitation maximumof the Tdap is 430 nm and of the PcSi vinyl is 350 nm. The emissionmaximum of the Tdap is 650 nm. The table below shows the results.

    ______________________________________                                                                   Fluorescence                                                                  Intensity at                                       Tdap/PcSi vinyl                                                                              Excitation λ (nm)                                                                  680 nm                                             ______________________________________                                        1/1            350         490                                                1/1            430         83                                                 1/1            450         38                                                 1/1            470         11                                                 2/1            350         580                                                2/1            430         830                                                2/1            450         460                                                2/1            470         220                                                6/1            350         600                                                6/1            430         1800                                               6/1            450         800                                                6/1            470         200                                                ______________________________________                                    

These results show that as the molar ratio of donor to acceptor in thelatex particles increases from 1/1 to 6/1, the energy transfer, asmeasured by the fluorescence intensity of the acceptor dye, becomessignificantly more efficient. There was no observable emission of theTdap dye in the particles at the emission maximum of 650 nm suggestingthat the energy transfer is very efficient. The data indicates that thefluorescence intensity of the latex particles, generated through anenergy transfer pathway, is affected by the "light gathering" capabilityof the donor dye. Thus, optimization of the fluorescence intensity ofthe latex particles should involve changing the molar ratio of donor toacceptor.

EXAMPLE 14 Effect of Incorporation of Different Dyes on Ouenching andFluorescence Intensity of Latex Particles

Five different silicon phthalocyanines, synthesized as described inexamples 2-6, were incorporated into 67 nm surfactant-free, polystyrenelatex particles (Interfacial Dynamics Corp. Inc. Portland, Oreg.) insets of 1, 3 or 5 dyes according to the following methods. Each siliconphthalocyanine derivative had maximum excitation and emissionwavelengths at 350 nm and 680 nm, respectively. After preparation ofeach dye-latex, each suspension was diluted to 0.057% solids in eitherwater or tetrahydrofuran. The dye-latex solutions were excited at 350 nmand the fluorescence intensity at 680 nm was measured. The intensity offluorescence in water divided by the intensity of fluorescence intetrahydrofuran minus 1 is the degree of quenching of the dyes in thelatex particles.

Preparation of One Phthalocyanine Dye in Latex

A solution of PcSi pentafluoro dye (0.02 mg) in tetrahydrofuran (0.1 mL)was added dropwise over 5 minutes to a stirred 2% solids solution oflatex particles (1.0 mL). The latex suspension was stirred at roomtemperature for 6 hours, then transferred to dialysis tubing(Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston,Tex.) and the dye-latex solution was dialyzed against water for 12-15hours at 4° C. The dye-latex solution was removed from dialysis and thesolids concentration was adjusted to 1.6%.

Preparation of Three Phthalocyanine Dyes in Latex

A solution which consists of PcSi pentafluoro, PcSi trihexyl and PcSicyano dyes in equimolar amounts to total 0.02 mg dye in tetrahydrofuran(0.1 mL), was added drop-wise over 5 minutes to a stirred 2% solidssolution of latex particles (1.0 mL). The latex suspension was stirredat room temperature for 6 hours, then transferred to dialysis tubing(Spectra-por, 12-14,000 molecular weight cutoff, Spectrum, Houston,Tex.) and the dye-latex solution was dialyzed against water for 12-15hours at 4° C. The dye-latex solution was removed from dialysis and thesolids concentration was adjusted to 1.6%.

Preparation of Five Phthalocyanine Dyes in Latex

A solution which consists of PcSi pentafluoro, PcSi trihexyl, PcSicyano, PcSi vinyl and PcSi methyl ester dyes in equimolar amounts tototal 0.02 mg dye in tetrahydrofuran (0.1 mL), was added dropwise over 5minutes to a stirred 2% solids solution of latex particles solution (1.0mL). The latex suspension was stirred at room temperature for 6 hours,then transferred to dialysis tubing (Spectra-por, 12-14,000 molecularweight cutoff, Spectrum, Houston, Tex.) and the dye-latex solution wasdialyzed against water for 12-15 hours at 4° C. The dye-latex solutionswere removed from dialysis and the % solids concentration was adjustedto 1.6%.

The table that follows illustrates the results of the fluorescenceexperiments.

    ______________________________________                                        Dyes Entrapped Intensity                                                                              % Quenching                                           ______________________________________                                        1              413      72                                                    3              561      56                                                    5              747      49                                                    ______________________________________                                    

The data show that as the number of different dyes entrapped into thelatex goes from 1 to 3 to 5, the fluorescence intensity increasesbecause the quenching in the particles decreases.

EXAMPLE 15 Preparation and Characterization of Fluorescence EnergyTransfer Dye Latex (Table 1) and Fluorescent Latex Incorporating HybridPhthalocyanine Derivatives (Table 2)

A variety of fluorescent energy transfer latexes were prepared withvarious donor and acceptor dye molecules. Table 1 shows the loadingconcentrations of the respective donor and acceptor dyes, the mole ratioof the donor and acceptor dyes, the dye loading solvent system asdescribed in Example 10 and the excitation and emission wavelengths andthe fluorescence intensity for each particle size at the specifiedsolids concentration. For some of the energy transfer latexes, the samedye pair was incorporated into different diameter latexes. Thefluorescence energy transfer efficiency of the entries is greater than80%. The dye system represented in line 56 is a fluorescence energytransfer compound (FET compound) so that the donor and acceptor pairreside in the molecule before incorporation into latex.

Table 2 shows the characteristics of latex particles incorporated withhybrid phthalocyanine derivatives as described in Example 10 and thefluorescence intensity at the specified solids concentration.

                                      TABLE 1                                     __________________________________________________________________________                                           MOLE  SOLVENT                                          LOADING          LOADING                                                                             DONOR:                                                                              SYSTEM      EMISSION                             CONC.            CONC. MOLE  (LATEX                                                                              INTENSITY                                                                           MAXIMUM              DONOR DYE       (mg/mL)                                                                             ACCEPTOR DYE                                                                             (mg/mL)                                                                             ACCEPTOR                                                                            SIZE) (% SOLID)                                                                           (EXCIT.)             __________________________________________________________________________    1. trans-4- 4-(Dibutyl amino)                                                                 0.12  Silicon phthalocyanine                                                                   0.1   2:1   THF   340   679 nm               styryl!-1-methyl pyridinium iodide                                                            mg/mL bis(dimethylvinylsilyl-                                                                  mg/mL       (0.067                                                                              (0.0019%)                                                                           (475 nm)                                   oxide)                 μm)                           2. trans-4- 4-(Dibutyl amino)                                                                 0.1   Silicon 2,3-naphthalo-                                                                   0.23  1:1   DMF   347   789 nm               styryl!-1-methyl pyridinium iodide                                                            mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (475 nm)                                   vinylsilyloxide)       μm)                           3. trans-4- 4-(Dibutyl amino)                                                                 0.1   1,1'-Dihexyl-3,3,3',3'-                                                                  0.144 1:1   DMF   688   688 nm               styryl!-1-methyl pyridinium iodide                                                            mg/mL tetramethylindodicarbo-                                                                  mg/mL       (0.067                                                                              (0.057%)                                                                            (645 nm)                                   cyanine iodide         μm)                           4. Meso-tetra-2-aminophenyl                                                                   0.18  Silicon phthalocyanine                                                                   0.1   2:1   THF   1000  679 nm               porphine        mg/mL bis(dimethylvinylsilyl-                                                                  mg/mL       (0.202                                                                              (0.00095%)                                                                          (420 nm)                                   oxide)                 μm)                           5. Meso-tetra-2-aminophenyl                                                                   0.1   1,1'-Dihexyl-3,3,3',3'-                                                                  0.098 1:1   DMF   157   676 nm               porphine        mg/mL tetramethylindodicarbo-                                                                  mg/mL       (0.067                                                                              (0.0019%)                                                                           (645 nm)                                   cyanine iodide         μm)                           6. Meso-tetra-2-                                                                              0.21  Silicon phthalocyanine                                                                   0.1   2:1   THF   209   679 nm               dimethylaminophenyl porphine                                                                  mg/mL bis(dimethylvinyl-                                                                       mg/mL       (0.412                                                                              (0.00095%)                                                                          (430 nm)                                   silyloxide)            μm)                           7. 3-Ethyl-3'-ethyl carboxyethyl-                                                             0.056 Silicon 2,3-naphthalo-                                                                   0.25  4:1   DMF   289   785 nm               thiadicarbocyanine iodide                                                                     mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)                                   vinylsilyloxide)       μm)                           8. 1,1'-Dioctadecyl-3,3,3,3',3'-                                                              0.036 Silicon 2,3-naphthalo-                                                                   0.013 4:1   DMF   324   787 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)             perchlorate           vinylsilyloxide)       μm)                           9. 1,1'-Diethyl-3,3,3',3'-                                                                    0.078 Silicon 2,3-naphthalo-                                                                   0.025 6:1   DMF   723   787 nm               tetramethylindodi-carbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (635 nm)             iodide                vinylsilyloxide)       μm)                           10. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.094 Silicon 2,3-naphthalo-                                                                   0.025 6:1   DMF   907   783 nm               tetramethylindodicarbocyanine                                                                 mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (635 nm)             iodide                vinylsilyloxide)       μm)                           11. 3,3'-Diethyl                                                                              0.013 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   12    788 nm               thiadicarbocyanine iodide                                                                     mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)                                   vinylsilyloxide)       μm)                           12. 3,3'-Dipropyl                                                                             0.013 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   65    788 nm               thiadicarbocyanine iodide                                                                     mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (660 nm)                                   vinylsilyloxide)       μm)                           13. 1,9-Dimethyl-methylene                                                                    0.008 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   57    788 nm               blue, chloride  mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)                                   vinylsilyloxide)       μm)                           14. N,N'-Di(3-trimethyl-                                                                      0.013 Silicon 2,3-                                                                             0.025 1:1   DMF   63    788 nm               ammoniumpropyl) thia-                                                                         mg/mL naphthalocyanine                                                                         mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)             dicarbocyanine tribromide                                                                           bis(dimethylvinyl-     μm)                                                 silyloxide)                                             15. 1,1',3,3,3',3'-Hexamethyl-                                                                0.012 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   33    788 nm               indo-tricarbocyanine perchlorate                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)                                   vinylsilyloxide)       μm)                           16. N-(3-Triethyl-                                                                            0.014 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   55    788 nm               ammoniumpropyl)-4-(4-(p-                                                                      mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (500 nm)             dibutylaminophenyl) butadienyl)                                                                     vinylsilyloxide)       μm)                           pyridium, dibromide                                                           17. 1,1',3,3,3',3'-Hexamethyl-                                                                0.015 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   8     788 nm               4,4'-5,5'-dibenzo-2,2'-indo-                                                                  mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)             tricarbocyanine perchlorate                                                                         vinylsilyloxide)       μm)                           18. Fluoroscein 0.264 Silicon phthalocyanine                                                                   0.1   6:1   THF   517   683 nm                               mg/mL bis(dimethylvinyl-                                                                       mg/mL       (0.067                                                                              (0.057%)                                                                            (485 nm)                                   silyloxide)            μm)                           19. Chlorophyll B                                                                             0.087 Silicon 2,3-naphthalo-                                                                   0.025 4:1   THF   72    783 nm                               mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (440 nm)                                   vinylsilyloxide)       μm)                           20. Chlorophyll B                                                                             0.244 Silicon phthalocyanine                                                                   0.1   2:1   THF   140   679 nm                               mg/mL bis(dimethylvinyl-                                                                       mg/mL       (0.067                                                                              (0.0019%)                                                                           (440 nm)                                   silyloxide)            μm)                           21. trans-4- 4-(Dibutyl                                                                       0.181 Silicon phthalocyanine                                                                   0.07  4:1:1 THF   300   681 nm               amino)styryl!-1-methyl pyridinium                                                             mg/mL bis(dimethylpenta-                                                                       mg/mL       (0.067                                                                              (0.0019%)                                                                           (475 nm)             iodide                fluorophenylsilyloxide)                                                                              μm)                                                 +                                                                             Silicon phthalocyanine                                                                   0.05                                                               bis(dimethylvinylsilyl-                                                                  mg/mL                                                              oxide)                                                  22. trans-4- 4-(Dibutyl amino)                                                                0.072 Silicon phthalocyanine                                                                   0.04  4:1:1:1                                                                             THF   206   681 nm               styryl!-1 -methyl pyridinium iodide                                                           mg/mL bis(trihexylsilyloxide)                                                                  mg/mL       (0.067                                                                              (0.0019%)                                                                           (475 nm)                                   +                      μm)                                                 Silicon phthalocyanine                                                                   0.04                                                               bis(dimethylpentafluoro-                                                                 mg/mL                                                              phenylsilyloxide)                                                             +                                                                             Silicon phthalocyanine                                                                   0.03                                                               bis(dimethylvinylsilyl-                                                                  mg/mL                                                              oxide)                                                  23. 3-Ethyl-3'-carboxyethylthia-                                                              0.013 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   76    788 nm               dicarbocyanine iodide                                                                         mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (625 nm)                                   vinylsilyloxide)       μm)                           24. 3-Ethyl-3'-ethyl-carboxy-                                                                 0.013 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   135   788 nm               ethyloxathiadicarbocyanine iodide                                                             mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (630 nm)                                   vinylsilyloxide)       μm)                           25. 3,3'-Diethylthia-                                                                         0.013 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   59    787 nm               dicarbocyanine iodide                                                                         mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (660 nm)                                   vinylsilyloxide)       μm)                           26. 3,3'-Diethyloxa-                                                                          0.012 Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   57    787 nm               dicarbocyanine iodide                                                                         mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (590 nm)                                   vinylsilyloxide)       μm)                           27. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.094 Silicon 2,3-naphthalo-                                                                   0.025 6:1:2 DMF   127   788 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.057%)                                                                            (650 nm)             iodide                vinylsilyloxide)       μm CML)                                             +                                                                             Silicon naphthalocyanine                                                                 0.05                                                               bis(dimethylethyl-                                                                       mg/mL                                                              maleimidosilyloxide)                                    28. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.094 Silicon 2,3-naphthalo-                                                                   0.025 6:1:2 DMF   193   788 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.057%)                                                                            (635 nm)             iodide                vinylsilyloxide)       μm CML)                                             +                                                                             Silicon phthalocyanine                                                                   0.05                                                               bis(dimethylethyl-                                                                       mg/mL                                                              maleimidosilyloxide)                                    29. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.03  Silicon 2,3-naphthalo-                                                                   0.05  1:1   DMF   275   788 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.057%)                                                                            (650 nm)             iodide                hexylvinylsilyloxide)  μm CML)                       30. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.1   Silicon 2,3 naphthalo-                                                                   0.2   1:1   DMF   163   798 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.057%)                                                                            (650 nm)             iodide                triphenylsilyloxide)   μm CML)                       31. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.09  Silicon naphthalocyanine                                                                 0.05  4:1   DMF   153   790 nm               tetramethyl-indodicarbocyanine                                                                mg/mL bis(dimethylretinol)                                                                     mg/mL       (0.431                                                                              (0.057%)                                                                            (650 nm)             iodide                                       μm CML)                       32. 1,1',3,3,3',3'-Hexamethyl-                                                                0.216 Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   0.4   788 nm               indotricarbocyanine perchlorate                                                               mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.00057%)                                                                          (635 nm)                                   vinylsilyloxide)       μm CML)                       33. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.512 1,1',3,3,3',3'-                                                                          0.1   4:1   DMF   0.9   776 nm               tetramethylindo-dicarbocyanine                                                                mg/mL Hexamethylindotri-                                                                       mg/mL       (0.431                                                                              (0.00057%)                                                                          (635 nm)             iodide                carbocyanine perchlorate                                                                             μm CML)                       34. Lithium tetraacetylide boron                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   22    788 nm               complex of 1,1'-Dihexyl-                                                                      mg/mL cyanine bis(di-methyl-                                                                   mg/mL       (0.216                                                                              (0.00057%)                                                                          (635 nm)             3,3,3',3'-tetramethylindo-                                                                          hexylvinylsilyloxide)  μm CML)                       dicarbocyanine iodide                                                         35. Silicon phthalocyanine                                                                    0.334 Silicon 2,3-naphthalo-                                                                   0.1   10:1  DMF   1     800 nm               bis(di-methylvinylsilyloxide)                                                                 mg/mL cyanine bis(di-methyl-                                                                   mg/mL       (0.216                                                                              (0.00057%)                                                                          (650 nm)                                   hexylvinylsilyloxide)  μm CML)                       36. 1,1',3,3,3',3'-                                                                           0.23  Silicon 2,3-naphthalo-                                                                   0.1   10:1  DMF   0.4   780 nm               Hexamethylindotri-carbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (635 nm)             perchlorate           hexylvinylsilyloxide)  μm CML)                       37. 1,1',3,3,3',3'-                                                                           0.19  Silicon octaethoxy 2,3-                                                                  0.1   10:1  DMF   0.7   780 nm               Hexamethylindotri-carbocyanine                                                                mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (635 nm)             perchlorate           methylhexylvinyl-      μm CML)                                             silyloxide)                                             38. Oxazine 1 perchlorate                                                                     0.01  Silicon 2,3-naphthalo-                                                                   0.025 1:1   DMF   291   788 nm                               mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.067                                                                              (0.057%)                                                                            (650 nm)                                   vinylsilyloxide)       μm)                           39. 3,3'-Dipropyl-                                                                            0.232 Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   0.4   788 nm               thiadicarbocyanine iodide                                                                     mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.431                                                                              (0.00057%)                                                                          (635 nm                                    vinylsilyloxide)       μm CML)                       40. Copper tetra-tert-butyl                                                                   0.72  Silicon 2,3-naphthalo-                                                                   0.1   1:1   DMF   0.2   788 nm               phthalocyanine  mg/mL cyanine bis(di-                                                                          mg/mL       (0.216                                                                              (0.00057%)                                                                          (650 nm)                                   methylhexylvinyl-      μm CML)                                             silyloxide)                                             41. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   42    785 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              hexylvinylsilyloxide)  μm CML)                       42. Aluminum tetra-tert-butyl                                                                 0.28  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   0.5   788 nm               phthalocyanine hydroxide                                                                      mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (650 nm)                                   hexylvinylsilyloxide)  μm CML)                       43. Aluminum    0.29  Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   0.1   788 nm               tetra-tert-butylphthalocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (650 nm)             chloride              hexylvinylsilyloxide)  μm CML)                       44. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.14  Aluminum octabutoxy-                                                                     0.1   4:1   THF   1.8   774 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL phthalocyanine                                                                           mg/mL       (0.216                                                                              (0.00057%)                                                                          (650 nm)             3a,4a-diazo-s-indacene                                                                              triethylsilyloxide     μm CML)                       45. Iron phthalocyanine                                                                       0.26  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   0.3   788 nm               bis(tert-butyl isocyanide)                                                                    mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   hexylvinylsilyloxide)  μm CML)                       46. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Octabutoxy-                                                                              0.1   4:1   THF   0.7   783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL phthalocyanine                                                                           mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                       μm CML)                       47. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.15  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   16.9  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              phenylpentafluoro-     μm CML)                                             silyloxide)                                             48. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.19  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   31.5  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              vinylsilyloxide)       μm CML)                       49. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.15  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   13.1  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              bis(diphenylvinyl-     μm CML)                                             silyloxide)                                             50. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.15  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   4.7   783                  butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              maleimidoethoxysilyl-  μm CML)                                             oxide)                                                  51. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.14  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   11.7  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              silyl-oxide-trans-stilbene)                                                                          μm CML)                       52. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.12  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   22.3  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(tri-deca-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              fluoro-1,1,2,2-tetra-  μm CML)                                             hydrooctyl-1-dimethyl-                                                        silyloxide)                                             53. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.12  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   16.1  783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              retinol)               μm CML)                       54. Germanium tetra-tert-butyl                                                                0.3   Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   1.3   783 nm               phthalocyanine dihydroxide                                                                    mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   hexylvinylsilyloxide)  μm CML)                       55. Germanium tetra-tert-butyl                                                                0.3   Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   0.6   783 nm               phthalocyanine dichloride                                                                     mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)5                                  hexylvinylsilyloxide)  μm CML)                       56. Silicon phthalocyanine bis                                                                0.15  Silicon phthalocyanine THF   209   681 nm               (maleimide-fluoroscein)                                                                       mg/mL bis (maleimide-        (0.067                                                                              (0.0019%)                                                                           (470 nm)             FET COMPOUND          fluoroscein)           μm)                                                 FET COMPOUND                                            57. 3,3'-Diethylthia-                                                                         0.57  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   DMF   -0.048                                                                              832 nm               tricarbocyanine iodide                                                                        mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           iodide                                                  58. 1,1',3,3,3',3'-                                                                           0.61  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   DMF   -0.149                                                                              832 nm               Hexamethylindotri-carbocyanine                                                                mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             perchlorate           diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           iodide                                                  59. 1,1',3,3,3',3'-Hexamethyl-                                                                0.51  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   DMF   -0.046                                                                              832 nm               4,4',5,5'-dibenzo-2,2'-indo-                                                                  mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             tricarbocyanine perchlorate                                                                         diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           iodide                                                  60. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.23  Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   -14.12                                                                              783 nm               tetramethyl-indodicarbocyanine                                                                mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             iodide                hexylvinylsilyloxide)  μm CML)                       61. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.1   4:1   DMF   -5.00                                                                               783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              hexylvinylsilyloxide)  μm CML)                       62. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.26  Silicon octaethoxy2,3-                                                                   0.1   4:1   DMF   -2.74                                                                               858 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              methylhexylvinylsilyl- μm CML)                                             oxide)                                                  63. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.32  Octabutoxy-                                                                              0.1   4:1   DMF   -4.07                                                                               762 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL phthalocyanine                                                                           mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                       μm CML)                       64. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.28  Octabutoxy-                                                                              0.1   4:1   DMF   -1.76                                                                               772 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                       μm CML)                       65. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.19  Silicon octaethoxy2,3-                                                                   0.1   4:1   DMF   -0.712                                                                              858 nm               tetramethylindo-dicarbocyanine                                                                mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             iodide                methylhexylvinyl-      μm CML)                                             silyloxide)                                             66. 3,3'-Diethylthia-                                                                         0.16  Silicon octaethoxy2,3-                                                                   0.1   4:1   DMF   -0.058                                                                              858 nm               tricarbocyanine iodide                                                                        mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   methylhexylvinyl-      μm CML)                                             silyloxide)                                             67. 1,1',3,3,3',3'-                                                                           0.15  Silicon octaethoxy 2,3-                                                                  0.1   4:1   DMF   -0.141                                                                              858 nm               Hexamethylindotri-carbocyanine                                                                mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             perchlorate           methylhexylvinyl-      μm CML)                                             silyloxide)                                             68. 1,1',3,3,3',3'-Hexamethyl-                                                                0.19  Silicon octaethoxy 2,3-                                                                  0.1   4:1   DMF   -0.058                                                                              858 nm               4,4',5,5'-dibenzo-2,2'-indo-                                                                  mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             tricarbocyanine perchlorate                                                                         methylhexylvinyl-      μm CML)                                             silyloxide)                                             69. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.2   Silicon octaethoxy 2,3-                                                                  0.15  4:1   THF   -2.720                                                                              858 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL naphthalocyanine bis(di-                                                                 mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              methylhexylvinyl-      μm CML)                                             silyloxide)                                             70. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.1   4:1:1 THF   -2.38                                                                               858 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              hexylvinylsilyloxide) +                                                                              μm CML)                                             Silicon octaethoxy 2,3-                                                       naphthalocyanine bis(di-                                                                 0. 12                                                              methylhexylvinylsilyl-                                                                   mg/mL                                                              oxide)                                                  71. Silicon phthalocyanine                                                                    0.36  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -8.10                                                                               832 nm               bis(di-methylvinylsilyloxide)                                                                 mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             72. Tetrakis(4-cumyl-phenoxy)                                                                 0.48  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   -0.397                                                                              783 nm               phthalocyanine  mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   hexylvinylsilyloxide)  μm CML)                       73. Tetrakis(4-cumyl-phenoxy)                                                                 0.68  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -0.128                                                                              832 nm               phthalocyanine  mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             74. Tetrakis(phenylthio)                                                                      0.34  Silicon 2,3-naphthalo-                                                                   0.1   4:1   THF   -0.374                                                                              788 nm               phthalocyanine  mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   hexylvinylsilyloxide)  μm CML)                       75. Tetrakis(phenylthio)                                                                      0.28  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -0.109                                                                              832 nm               phthalocyanine  mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             76. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.24  Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -1.724                                                                              >900 nm              butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              dichloride             μm CML)                       77. Tetrakis    0.36  Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.162                                                                              >900 nm              (4-cumylphenoxy) phthalocyanine                                                               mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   dichloride             μm CML)                       78. Tetrakis(phenylthio)                                                                      0.26  Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.061                                                                              >900 nm              phthalocyanine  mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   dichloride             μm CML)                       79. Germanium tetra-tert-butyl                                                                0.42  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -0.109                                                                              >900 nm              phthalocyanine dihydroxide                                                                    mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             80. Germanium tetra-tert-butyl                                                                0.22  Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.045                                                                              >900 nm              phthalocyanine dihydroxide                                                                    mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   dichloride             μm CML)                       81. Germanium tetra-tert-butyl                                                                0.2   Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.042                                                                              >900 nm              phthalocyanine dihydroxide                                                                    mg/mL naphthalocyanine bis                                                                     mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   (triethylsilyloxide)   μm CML)                       82. Germanium tetra-tert-butyl                                                                0.42  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -0.081                                                                              832 nm               phthalocyanine dichloride                                                                     mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             83. Germanium tetra-tert-butyl                                                                0.22  Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.052                                                                              >900 nm              phthalocyanine dichloride                                                                     mg/mL naphthalocyanine                                                                         mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   dichloride             μm CML)                       84. Germanium tetra-tert-butyl                                                                0.2   Tin octabutoxy 2,3-                                                                      0.1   4:1   THF   -0.050                                                                              >900 nm              phthalocyanine dichloride                                                                     mg/mL naphthalocyanine bis                                                                     mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)                                   (triethylsilyloxide)   μm CML)                       85. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.1   4:1:1 THF   -0.315                                                                              858 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              hexylvinylsilyloxide) +                                                                              μm CML)                                             5,5'-Dichloro-1,1'-                                                           diphenylamino-3,3'-                                                                      0.072                                                              diethyl-10,12-ethylene-                                                                  mg/mL                                                              thiatricarbocyanine                                                           perchlorate                                             86. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.24  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -2.230                                                                              832 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             87. 1,1'-Dihexyl-3,3,3',3'-                                                                   0.34  5,5'-Dichloro-1,1'-                                                                      0.1   4:1   THF   -0.545                                                                              823 nm               tetramethyl-indodicarbocyanine                                                                mg/mL diphenylamino-3,3'-                                                                      mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             iodide                diethyl-10,12-ethylene-                                                                              μm CML)                                             thiatricarbocyanine                                                           perchlorate                                             88. (E,E)-3,5-bis-(4-phenyl-1,3-                                                              0.16  Silicon 2,3-naphthalo-                                                                   0.07  4:1:1 THF   49    783 nm               butadienyl)-4,4-difluoro-4-bora-                                                              mg/mL cyanine bis(dimethyl-                                                                    mg/mL       (0.216                                                                              (0.00057%)                                                                          (670 nm)             3a,4a-diazo-s-indacene                                                                              hexylvinylsilyloxide) +                                                                              μm CML)                                             Silicon 2,3-naphthalo-                                                        cyanine bis(dimethyl-                                                                    0.07                                                               pentafluorophenyl-silyl-                                                                 mg/mL                                                              oxide)                                                  __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________                         LOADING                                                                       CONC. SOLVENT                                                                             LATEX            EMISSION                    HYBRID COMPOUND      (mg/mL)                                                                             SYSTEM                                                                              SIZE  % SOLID                                                                            INTENSITY                                                                           MAXIMUM                                                                             EXCITATION            __________________________________________________________________________    1. Silicon  di(1,6-diphenylnaphthalocyanine)!                                                      2.0   THF   0.216 0.00057%                                                                           50    760 nm                                                                              650 nm                diphthalocyanine bis(dimethylhexyl-                                                                mg/mL       μm CML                                    vinylsilyloxide)                                                              2. Silicon  di(1,6-diphenylnaphthalocyanine)!                                                      2.0   THF   0.216 0.00057%                                                                           0.7/0.5                                                                             765 nm/                                                                             650 nm                tetrafluorophthalocyanine                                                                          mg/mL       μm CML        825 nm                      phthalocyanine                                                                bis(dimethylhexylvinylsilyloxide)                                             3. Silicon  di(1,6-diphenylnaphthalocyanine)!                                                      1.5   THF   0.216 0.00057%                                                                           0.5/0.3                                                                             770 nm/                                                                             650 nm                tetrafluorophthalocyanine                                                                          mg/mL       μm CML        839 nm                      phthalocyanine                                                                bis(dimethylpentafluorophenylsilyloxide)                                      4. Silicon  di(1,6-diphenylnaphthalocyanine)!                                                      0.1   THF   0.216 0.00057%                                                                           0.2   775 nm                                                                              650 nm                diphthalocyanine bis(dimethylpentafluoro-                                                          mg/mL       μm CML                                    phenylsilyloxide)                                                             5. Silicon  di(1,6-diphenylnaphthalocyanine)!                                                      1.5   THF   0.216 0.00057%                                                                           19    758 nm                                                                              650 nm                di(tert-butyl-phthalocyanine) bis(dimethyl-                                                        mg/mL       μm CML                                    hexylvinylsilyloxide)                                                         __________________________________________________________________________

EXAMPLE 16 Adsorption of Anti-Human Chorionic Gonadotropin (hCG)Antibody to Latex Particles

A typical example of the adsorptions of an antibody to dyed latexparticles, prepared as described in Example 10, and of a complementaryantibody to undyed latex particles, both of which can be used in asandwich assay for hCG, is outlined below. Those skilled in the art willrecognize that various techniques are available to adsorb or tocovalently couple proteins, peptides, ligand analogues nucleotides andnucleic acids to latex particles. A solution of dye latex (0.1 mL, 2%solids, 412 nm; entry 10, Table 1) was added quickly while vortexing toa solution of anti-β hCG monoclonal antibody (0.2 mL, 6.6 mg/mL; AppliedBiotech Inc., San Diego, Calif.) in 20 mM sodium borate/150 mM sodiumchloride, pH 8.2. A solution of 0.1M potassium citrate, pH 3, (0.04 mL)was added quickly while vortexing to the antibody latex solution at roomtemperature and the pH of the resulting solution was 3.5. The solutionincubated at room temperature for 5 minutes, then a solution of 2Mpotassium borate, pH 9.7 (0.025 mL) was added quickly while vortexing tobring the pH to about 8.5. This latex antibody conjugate was dialyzed(Spectra-por dialysis tubing, molecular weight cutoff of 300,000,Spectrum, Houston, Tex.) against 4 changes of 2 L each of 20 mM sodiumborate/150 mM sodium chloride, pH 8.2 at 4° C. for 4 days. The dialyzedlatex conjugate was then removed from the dialysis tubing and the solidsconcentration was calculated to be 0.4%. This conjugate can be used forimmunoassays for hCG in serum. The latex has excitation and emissionwavelengths of 650 nm and 780 nm, respectively.

A solution of polystyrene sulfate latex (0.036 mL, 8.4% solids, 1000 nm;Interfacial Dynamics Corp., Inc., Portland Oreg.) was added quickly, atroom temperature, while vortexing to a solution consisting of anti-α hCGmonoclonal antibody (0.12 mL, 10.3 mg/mL; Applied Biotech Inc. SanDiego, Calif.) in 20 mM sodium borate/150 mM sodium chloride, pH 8.2 and0.1M potassium citrate, pH 3, (0.6 mL). The solution incubated at roomtemperature for 5 minutes and was subjected to centrifugation in anEppendorf centrifuge (2000×g for 5 min). The supernatant was removed,the pellet was resuspended in 0.1M potassium phosphate, pH 7, (1.5 mL)and the suspension was subjected to centrifugation as described above.This process was repeated 2 times more and in the final centrifugation,the pellet was resuspended with 0.1M potassium phosphate, pH 7 (0.3 mL)to make 1% solids. This antibody latex is used on a solid phase, such asa membrane, to capture the hCG-dye antibody latex conjugate complex in areaction mixture in an immunoassay for hCG.

EXAMPLE 17 Immunoassay for hCG

The solid phase anti-a hCG latex solution (0.005 mL, 1% solids; example16) can be applied to a 2 cm² piece of 0.45 micron nylon membrane(Millipore Corp., Boston, Mass.) which has been treated with a 2%solution of condensed milk to lower non-specific binding interactions.This membrane can be used as the solid phase onto which is captured thehCG dye latex conjugate complex. Thus, an hCG assay can be performed byaddition of dye latex conjugate (0.025 mL, example 16) to 0.1 mL samplesof serum suspected of containing hCG and also to 0.1 mL serum samplescontaining known amounts of hCG (10, 100, 300, 500 and 1000 mIU/mL). Theserum samples should be incubated about 10 minutes and then the samplesare applied to the solid phase membrane containing the solid phaselatex. The membrane should be placed over an absorbent so that the serumsample containing the dye latex conjugates flows through the solid phaselatex spot. After the serum solution has passed through the membrane,serum (0.5 mL) not containing the dye latex conjugate is applied to themembrane to remove unbound dye latex conjugate. The latex spots on themembranes are then placed in a front surface fluorescence accessory in afluorometer and the spot is excited at 650 nm and the fluorescenceintensity of the spot on each membrane is measured at 780 nm. Thefluorescence intensity as a function of the hCG concentrations of theknown samples is plotted. The fluorescence intensities of the unknownhCG serum samples can be compared to the known hCG concentrations fromthe graph.

EXAMPLE 18 Fluorometer for Measuring Near Infrared Emitting Dyes

The dye sample (2 mL sample volume in a 10 mm×10 mm quartz cuvette) wasexcited by a diode laser (Sun Laser SL-6; 1=670±10 nm, 0.95 mW) whichwas filtered by a low-pass cutoff filter (Corion LS700, passeswavelengths less than 700 nm). Fluorescence emission was detected at 90°to the incident diode laser beam. The emitted light was collected andfocused on a silicon photodiode (Melles Griot, Cat. #13DS1009) by acondenser consisting of two aspheric lenses (Melles Griot, Cat #01 LAG119). A high-pass cutoff filter (Schott Glass RG715) in front of theSilicon photodiode blocked scattered laser light at 670 nm but passedemitted light at wavelengths larger than 715 nm. The photocurrent fromthe silicon photodiode was amplified and displayed by a currentamplifier in nanoamps ("nA"), (Melles Griot, Cat. #13 AMP 003). In someinstances, 12 nm band filters were placed in front of the siliconphotodiode with center wavelengths at 730 nm, 790 nm, 850 nm, and 900nm.

EXAMPLE 19 Synthesis of Silicon 2,3-Naphthalocyaninebis(diphenylvinylsilyl-oxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (0.5 mL) containing diphenylvinylchlorosilane(28 μL, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred underargon at room temperature for 18 hours. The reaction mixture wasevaporated and the residue purified on a silica column equilibratingwith hexane and eluting the product with toluene as a long green band.The toluene fraction containing the product was evaporated to afford 5mg green solid.

EXAMPLE 20 Synthesis of Silicon 2,3-Naphthalocyaninebis(triphenylsilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing triphenylchlorosilane (37mg, 0.125 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argonat room temperature for 18 hours. The reaction mixture was evaporatedand the residue purified on a silica column equilibrating with hexaneand eluting the product with toluene as a green band. The toluenefraction containing the product was evaporated to afford 2.5 mg greensolid.

EXAMPLE 21 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylmaleimidoethoxysilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature for 18 hours. The reaction mixture was thentreated with N-(2-hydroxyethyl)maleimide (35 mg, 0.25 mmol) and stirredfor an additional 10 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane, thentoluene and eluting the product with toluene/10% isopropanol as a greenband. The eluate containing the product was evaporated to afford 3.5 mgof green solid.

EXAMPLE 22 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylsilyloxide-trans-stilbene)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature for 2 hours. The reaction mixture was thentreated with trans-4-hydroxystilbene (49 mg, 0.25 mmol) and stirred foran additional 5 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane andeluting the product with toluene as a long green band. The toluenefraction containing the product was evaporated to afford 4 mg greensolid.

EXAMPLE 23 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylhexylvinyl-silyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing7-oct-1-enyldimethylchlorosilane (32 μL, 0.125 mmol) and imidazole (7mg, 0.1 mmol) was stirred under argon at room temperature for 18 hours.The reaction mixture was evaporated and the residue purified on silicacolumn equilibrating with hexane and eluting the product with toluene asa green band. The toluene fraction containing the product was evaporatedand the residue treated with hexane to afford a dark green solid andlight green supernatant. The mixture was centrifuged, the supernatantremoved and the solid treated with more hexane and centrifuged. Thesupernatant was again removed and the solid dried under vacuum to yield7.3 mg of product.

EXAMPLE 24 Synthesis of Silicon 2,3-Naphthalocyaninebis(tridecafluoro-1,1,-2,2-tetrahydrooctyl-1-dimethylsilyloxide)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane (37 μL,0.1 mmol) and imidazole (7 mg, 0.1 mmol) was stirred under argon at roomtemperature for 2 hours. The reaction mixture was evaporated and theresidue purified on a silica column equilibrating with hexane andeluting with hexane/20% toluene followed by hexane/40% toluene to affordthe product as a green band. The product eluate was evaporated and theresidue treated with hexane to afford a green solid. The mixture wascentrifuged, the supernatant removed and the solid treated with morehexane and recentrifuged. The supernatant was again removed and thegreen solid dried under vacuum to yield 7.5 mg of product.

EXAMPLE 25 Synthesis of Silicon 2,3-Naphthalocyaninebis(dimethylretinol)

A suspension of silicon 2,3-naphthalocyanine dihydroxide (39 mg, 0.05mmol) in dimethylformamide (1 mL) containing dichlorodimethylsilane(13.5 μL, 0.11 mmol) and imidazole (14 mg, 0.2 mmol) was stirred underargon at room temperature. After 20 minutes, the reaction mixture wastreated with all-trans-retinol (72 mg, 0.25 mmol) and stirred for anadditional 1 hour. The reaction mixture was evaporated and the residuepurified on a silica column equilibrating with hexane and eluting theproduct with toluene as a long green band. The toluene fractioncontaining the product was evaporated and the residue treated withhexane to yield a dark green solid and light green supernatant. Themixture was centrifuged, the hexane removed and the solid dried undervacuum to yield 10 mg of final product.

EXAMPLE 26 Synthesis of Silicon Octaethoxy-2,3-naphthalocyanineDichloride

4,9-Diethoxy-1,3-diiminobenz f!isoindoline (0.6 g) was added under argonto freshly distilled quinoline (12 mL). After stirring for 10 minutes,silicon tetrachloride (4.0 mL) was added and the reaction mixture washeated at 190° C. for 1 hour. The reaction mixture was cooled to roomtemperature, and water (120 mL) was added slowly to hydrolyze theunreacted silicon tetrachloride. The blue-black precipitate was filteredoff and washed with methanol and acetone.

UV-vis (methylene chloride) (λ_(max) nm)): 768, 869.

EXAMPLE 27 Synthesis of Silicon Octaethoxy-2,3-NaphthalocyanineDihydroxide

A suspension of silicon octaethoxy-2,3-naphthalene dichloride (1.96 g)in pyridine (15 mL) containing water (15 mL) was refluxed for 18 hours.The suspension was cooled, the black precipitate filtered and washedwith water (10 mL). The precipitate was dried under vacuum and weighed(1.37 g, purple powder).

UV-vis (methylene chloride) (λ_(max) (nm)): 766, 867.

EXAMPLE 28 Synthesis of Silicon Octaethoxy-2,3-Naphthalocyaninebis(dimethylhexylvinylsilyloxide)

A suspension of silicon octaethoxy-2,3-naphthalene dihydroxide (1.0 g)in dimethylformamide (20 mL) containing 7-oct-1-enyldimethylchlorosilane(0.6 mL) and imidazole (140 mg) was stirred under argon at roomtemperature for 24 hours. The reaction mixture was evaporated with arotary evaporator, chromatographed (silica gel 70-230 mesh, 60 Å, 2×50cm, hexane-toluene(1:1)) vacuum dried, and weighed (46 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 855, 370000.

Infrared Spectrum(KBr): 3074, 2958, 2924, 2854, 1589, 1417, 1373, 1348,1262, 1238, 1194, 1161, 1111, 1044, 1025, 933, 909, 844, 799, 760 cm⁻¹.

¹ H-NMR (500 MHz, CDCl₃): δ9.0 (m, 2,5-Nc), 7.9 (m, 3,4-Nc), 5.3 (m,--CH₂), 4.6 (m, vinyl --CH₂), 3.5 (m, vinyl CH), 1.8 (m, --CH₃), 1.3 (m,ε --CH₂ ), 0.5 (m, δ --CH₂), 0.1 (m, γ --CH₂), -0.8 (m, β --CH₂), -1.7(m, α --CH₂), -2.3 (s, --CH₃).

EXAMPLE 29 Synthesis of Silicon Phthalocyaninebis(dimethylmaleimidofluorescein)

Fluorescein ATP (0.5 mg, 1.05 μmol) was treated with a solution of 0.12Mpotassium carbonate in 80% methanol (52 μL). After 5 minutes, thehydrolysis solution was quenched by the addition of 0.5M potassiumphosphate/0.1M potassium borate, pH 7.0 in 1N HCl (10 μL). The quenchedhydrolysis solution was evaporated to dryness, redissolved indimethylformamide (100 μL) and the resulting solution added to siliconphthalocyanine bis(dimethylmaleimidosilyloxide) in a 1.0 mL serum vial.The reaction mixture was then stirred at room temperature for 1 hour.The crude product was then chromatographed on two 3"×3" silica platesusing toluene/20% dimethylformamide. After elution, the plates weredried under vacuum and rechromatographed for a better separation. Theproduct band was scraped off, and treated with dimethylformamide (5 mL),vortexed 30 seconds and filtered from the silica. The filtrates wereevaporated to give 0.55 mg of greenish fluorescent solid.

EXAMPLE 30 Synthesis of Tin(IV) Octabutoxy-2,3-naphthalocyaninebis(triethylsilyloxide)

A mixture of triethylsilanol (77 μL), sodium (3.5 mg), and xylenes (5mL) was refluxed under argon for 1 hour and slightly cooled. A solutionof tin(IV) octabutoxy-2,3-naphthalocyanine dichloride (74 mg) in xylenes(5 mL) was added to the solution formed and the mixture was refluxed for20 minutes. The resultant was washed twice with water (25 mL each time),dried (MgSO₄), and evaporated to a dark red solid with a rotaryevaporator. This solid was chromatographed (silica gel 70-230 mesh, 60Å, 2×50 cm, toluene-isopropanol), vacuum dried, and weighed (17 mg).

UV-vis(tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 900, 174000.

EXAMPLE 31 Synthesis of Tin(IV) 2,3-Naphthalocyaninebis(triethylsilyloxide)

A mixture of triethylsilanol (77 μL), sodium (3.5 mg), and xylenes (8mL) was refluxed under argon for 1 hour and slightly cooled. Tin(IV)2,3-naphthalocyanine dichloride (45 mg) was added to the solutionformed, and the mixture was refluxed for 5 days. The suspension wasfiltered, and the solid was washed (xylenes and water), vacuum dried,and weighed (41 mg). The solid was chromatographed (silica gel 70-230mesh, 60 Å, 2×50 cm, methylene chloride-tetrahydrofuran), vacuum dried,and weighed (26 mg).

UV-vis(tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 700, 746; 786,253000.

Fluorescence (tetrahydrofuran) (λmax (nm)): 820.

EXAMPLE 32 Synthesis of Tin(IV) 2,3-Naphthalocyaninebis(dimethylhexylvinylsilyloxide)

A mixture of 7-oct-1-enyl dimethylsilanol (186 mg), sodium (7 mg), andxylenes (10 mL) was refluxed under argon for 4 hours and slightlycooled. Tin(IV) 2,3-naphthalocyanine dichloride (90 mg) was added to thesolution formed and the mixture was refluxed for 4 days. The suspensionwas filtered and the solid was washed with xylenes (5 mL) and water (5mL). The organic layer of the filtrate was separated, dried (MgSO₄), andevaporated with a rotary evaporator. The residue was triturated twicewith hexane (2 mL each time) to afford a bright green solid which wasvacuum dried and weighed (8.5 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm¹)): 670, 7200; 732,69900; 786, 84900.

EXAMPLE 33 Synthesis of Tin (IV) Octabutoxy-2,3-naphthalocyanineDichloride

Tin tetrachloride (234 μL) was added to a mixture ofoctabutoxy-2,3-naphthalocyanine (310 mg) in dry dimethylformamide (15mL) under an argon atmosphere and the mixture refluxed with stirring for6 hours. The resultant was allowed to cool, the suspension was filtered,and the dark red solid was washed with dimethylformamide (5 mL) andwater (5 mL), vacuum dried and weighed (288 mg).

EXAMPLE 34 Synthesis of Tin(IV) Octabutoxy-2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide)

A mixture of 7-oct-1-enyl dimethylsilanol (186 mg), sodium (7 mg), andxylenes (10 mL) was refluxed under argon for 5 hours and slightlycooled. Tin(IV) octabutoxy-2,3-naphthalocyanine dichloride (37 mg) wasadded to the solution formed, and the mixture was refluxed for 2 days.The resultant was washed with water (10 mL), dried (MgSO₄), andevaporated to a dark red solid with a rotary evaporator. This solid waschromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm,toluene-isopropanol), vacuum dried, and weighed (17 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 785; 893, 227000.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 789.

EXAMPLE 35 Synthesis of 7-Oct-1-enyl Dimethylsilanol

A solution of 7-oct-1-enyl dimethylchlorosilane (2.56 mL) in ether (2mL) was added dropwise over 1 hour to a stirring mixture oftriethylamine (1.5 mL), water (0.18 mL) and ether (15 mL) in anice/water bath. The resultant was stirred a further 1 hour in theice/water bath and filtered washing the filtered solid with ether (10mL). The filtrate was evaporated with a rotary evaporator and theresidue partitioned between hexane (30 mL) and water (30 mL). Theorganic layer was separated, dried (MgSO₄) and filtered through silicagel (70-230 mesh, 60 Å), washing with hexane (100 mL). The filtrate wasevaporated with a rotary evaporator to afford a colorless oil which wasvacuum dried and weighed (1.06 g).

EXAMPLE 36 Synthesis of Tetrabromotetrabutoxy-2,3-naphthalocyanine

1,4-dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) and2,3-dibromo-6,7-dicyanonaphthalene (168 mg) were added to a refluxingsolution of lithium metal (35 mg) in 1-butanol (2 mL) under an argonatmosphere. The reaction solution was maintained at reflux for 2 hours,cooled, and stirred into glacial acetic acid (10 mL). After 30 minutes,the solvent was evaporated with a rotary evaporator and the residuedissolved in methylene chloride (10 mL). The solution was washed twicewith 1N hydrochloric acid (10 mL each time), water (10 mL), dried(MgSO₄) and evaporated with a rotary evaporator. The residue waschromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene),the solid product triturated with hexane (2 mL), vacuum dried, andweighed (8 mg).

UV-vis(tetrahydrofuran) (λ_(max) (nm)) 743; 839.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 789.

EXAMPLE 37 Synthesis of Di(1,6-dibutoxy-2,3-naphthalocyanine)di(tetrafluorophthalocyanine)

1,4-Dibutoxynaphthalene-2,3-dicarbonitrile (161 mg) andtetrafluorophthalonitrile (100 mg) were added to a refluxing solution oflithium metal (35 mg) in 1-butanol (2 mL) under an argon atmosphere. Thereaction solution was maintained at reflux for 1 hour, cooled, andstirred into glacial acetic acid (10 mL). After 30 minutes the solventwas evaporated with a rotary evaporator and the residue dissolved inmethylene chloride (10 mL). The solution was washed twice with 1Nhydrochloric acid (10 mL each time), water (10 mL), dried (MgSO₄) andevaporated with a rotary evaporator. The residue was chromatographedtwice (silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene), thebright green fraction vacuum dried and weighed (10 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 679, 25800; 752,88200; 789, 76500.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 815.

EXAMPLE 38 Synthesis of Di(1,6-diphenyl-2,3-naphthalocyanine)di(tetrafluorophthalocyanine)

1,4-diphenylnaphthalene-2,3-dicarbonitrile (165 mg) andtetrafluorophthalonitrile (100 mg) were added to a refluxing solution oflithium metal (35 mg) in 1-butanol (2 mL) under an argon atmosphere. Thereaction solution was maintained at reflux for 1.5 hours, cooled, andstirred into glacial acetic acid (10 mL). After 30 minutes, the solventwas evaporated with a rotary evaporator and the residue dissolved inmethylene chloride (10 mL) The solution was washed twice with 1Nhydrochloric acid (10 mL each time), water (10 mL), dried (MgSO₄), andevaporated with a rotary evaporator. The residue was chromatographed(silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene), the brightgreen fraction vacuum dried and weighed (7 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 747, 86800.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 760.

EXAMPLE 39 Synthesis of Dibutoxy-1,3-diiminobenz f!isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-dibutoxynaphthalene-2,3-dicarbonitrile (1.61 g), 25% sodiummethoxide in methanol (1.14 mL), and dry 1-butanol (10 mL) for 30minutes. With continued ammonia introduction, the mixture was refluxedfor 30 minutes. After the resultant had cooled, the solvent was removedunder vacuum with a rotary evaporator. The residue was chromatographed(silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene-isopropanol) andthe yellow product treated with ether (10 mL), collected by filtration,washed with ether (10 mL), vacuum dried and weighed (517 mg).

¹ H-NMR (500 MHz, CDCl₃) δ8.22 (m, 5,8 --H), 7.65 (m, 6,7 --H), 4.23 (m,γ --CH₂), 1.97 (m, β --CH₂), 1.61 (m, α --CH₂), 1.04 (t, --CH₃).

EXAMPLE 40 Synthesis of Diethoxy-1,3-diiminobenz f!isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diethoxynaphthalene-2,3-dicarbonitrile (1.33 g), 25% sodiummethoxide in methanol (1.14 mL), and dry ethanol (10 mL) for 20 minutes.With continued ammonia introduction, the mixture was refluxed for 2hours. After the resultant had cooled, the solvent was removed undervacuum with a rotary evaporator. The residue was treated with methylenechloride (10 mL) and the product was collected by filtration, washedwith water (5 mL), methylene chloride (5 mL), vacuum dried and weighed(766 mg).

EXAMPLE 41 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!Diphthalocyanine Dihydroxide

Silicon tetrachloride (231 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz f!isoindoline (470 mg) and1,3-diiminoisoindoline (97 mg) in freshly distilled quinoline (5 mL)under an argon atmosphere and the mixture heated with stirring at 200°C. for 40 minutes. The resultant was allowed to cool slightly, treatedwith water (5 mL) and refluxed for 5 minutes. The mixture was cooled,treated with ether (30 mL) and filtered washing the solid with ether (10mL) and water (10 mL). The organic layer of the filtrate (which was darkgreen) was separated, washed with water (15 mL), dried (MgSO₄) andevaporated with a rotary evaporator. The residue was chromatographedthree times (silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane-methylenechloride), vacuum dried and weighed (55.5 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 640; 680; 714,67900; 742.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 750.

EXAMPLE 42 Synthesis of Silicon di(1,6-diethoxy-2,3-naphthalocyanine)!Diphthalocyanine Dihydroxide

Silicon tetrachloride (137 μL) was added to a mixture ofdiethoxy-1,3-diiminobenz f!isoindoline (227 mg) and1,3-diiminoisoindoline (58 mg) in freshly distilled quinoline (3 mL)under an argon atmosphere and the mixture heated with stirring at 200 °C. for two hours. The resultant was allowed to cool slightly, treatedwith water (3 mL) and refluxed for 5 minutes. The mixture was cooled,treated with ether (10 mL), and the dark blue solid product filteredoff, washed with ether (10 mL) and water (10 mL), vacuum dried andweighed (175 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 600, 632, 666, 700, 724, 788.

EXAMPLE 43 Synthesis of Silicon di(1,6-diethoxy-2,3-naphthalocyanine)!Diphthalocyanine bis(dimethylhexylvinylsilyloxide)

A mixture of silicon di(1,6-diethoxy-2,3-naphthalocyanine)!diphthalocyanine dihydroxide (85 mg), 7-oct-1-enyl dimethylchlorosilane(256 μL), imidazole (68 mg), and dimethylformamide (2 mL) was stirred atroom temperature for 24 hours. The resultant was concentrated undervacuum with a rotary evaporator. The residue was chromatographed (silicagel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene-isopropanol), vacuumdried, and weighed (32 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 601, 633, 667, 702, 731, 822,904.

EXAMPLE 44 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!Diphthalocyanine bis(dimethylhexylvinylsilyloxide) (FIG. 9).

A mixture of silicon di(1,6-diphenyl-2,3-naphthalocyanine)!diphthalocyanine dihydroxide (30 mg), 7-oct-1-enyl dimethylchlorosilane(115 μL), imidazole (30 mg) and dimethylformamide (650 μL) was stirredat room temperature for 30 minutes. The resultant was concentrated undervacuum on the rotary evaporator. The residue was chromatographed (silicagel 70-230 mesh, 60 Å, 2×50 cm, hexane-toluene), vacuum dried andweighed (38 mg).

¹ H-NMR (500 MHz, CDCl₃) δ8.31, 8.25 (m, 2,5-Nc, 10,13-Nc), 7.94 (m,Ar--Nc), 7.95, 7.74 (3,4-Nc, 11,12-Pc), 0.68 (m, ε --CH₂), 0.21 (m, δ--CH₂), -0.11 (m, γ --CH₂), -1.22 (nm, β --CH₂), -2.14 (m, α --CH₂),-2.76 (s, --CH₃).

UV-vis(tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 644; 684; 718,81100; 748.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 754.

EXAMPLE 45 Synthesis of Tetrafluoro-1,3-diiminobenz f!isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture oftetrafluorophthalonitrile (2.0 g), 25% sodium methoxide in methanol (2.3mL), and dry 1-butanol (10 mL) for 20 minutes. With continued ammoniaintroduction, the mixture was refluxed for 1 hour. After the resultanthad cooled, the solvent was removed under vacuum with a rotaryevaporator. The residue was treated with ether (50 mL) and the productwas collected by filtration, washed with water (10 mL), ether (10 mL),vacuum dried and weighed (0.45 g).

EXAMPLE 46 Synthesis of Diphenyl-1,3-diiminobenz f!isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diphenylnaphthalene-2,3-dicarbonitrile (4.3 g), 25% sodium methoxidein methanol (3.0 mL), and dry 1-butanol (25 mL) for 30 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 1.5 hours.After the resultant had cooled, the solvent was removed under vacuumwith a rotary evaporator. The residue was treated with methylenechloride (50 mL) and the product was collected by filtration, washedwith water (10 mL), methylene chloride (10 mL), vacuum dried and weighed(3.68 g).

EXAMPLE 47 Synthesis of Silicon di(1.6-diphenyl-2,3-naphthalocyanine)!di(tetrafluorophthalocyanine) Dihydroxide

Silicon tetrachloride (86 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz f!isoindoline (174 mg) andtetrafluoro-1,3-diiminoisoindoline (54 mg) in freshly distilledquinoline (1 mL) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1 hour. The resultant was allowed to coolslightly, treated with water (1 mL) and refluxed for 5 minutes. Themixture was cooled, treated with ether (10 mL) and filtered washing thesolid with water (2 mL) and ether (5 mL). The organic layer of thefiltrate was separated, washed with water (5 mL), dried (MgSO₄) andevaporated with a rotary evaporator.

The residue was chromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm,methylene chloride), vacuum dried and weighed (18 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻)): 727, 759, 809, 835.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 685, 760, 840.

EXAMPLE 48 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(1,6-diethoxyphthalocyanine) Phthalocyanine Dihydroxide

Silicon tetrachloride (172 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz f!isoindoline (347 mg),diethoxy-1,3-diiminobenz f!isoindoline (71 mg) and1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 mL)under an argon atmosphere and the mixture heated with stirring at 200 °C. for 1 hour. The resultant was allowed to cool slightly, treated withwater (2 mL) and refluxed for 5 minutes. The mixture was cooled, treatedwith ether (10 mL) and filtered washing the solid with water (5 mL) andether (5 mL). The organic layer of the filtrate was separated, washedwith water (10 mL), dried (MgSO₄) and evaporated with a rotaryevaporator. The residue was chromatographed (silica gel 70-230 mesh, 60Å, 2×50 cm, methylene chloride), vacuum dried and weighed (6 mg).

UV-vis (methylene chloride) (λ_(max) (nm)): 649, 693, 724, 758, 827.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 750.

EXAMPLE 49 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(tetrafluorophthalocyanine) Phthalocyanine Dihydroxide

Silicon tetrachloride (172 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz f!isoindoline (347 mg),tetrafluoro-1,3-diiminobenz f!isoindoline (54 mg) and1,3-diiminoisoindoline (36 mg) in freshly distilled quinoline (2 mL)under an argon atmosphere and the mixture heated with stirring at 200°C. for 1 hour. The resultant was allowed to cool slightly, treated withwater (2 mL) and refluxed for 5 minutes. The mixture was cooled, treatedwith ether (10 mL) and filtered washing the solid with water (5 mL) andether (5 mL). The organic layer of the filtrate was separated, washedwith water (10 mL), dried (MgSO₄) and evaporated with a rotaryevaporator. The residue was chromatographed (silica gel 70-230 mesh, 60Å, 2×50 cm, methylene chloride), vacuum dried and weighed (21 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 646, 689, 720, 753, 790.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 760.

EXAMPLE 50 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(tetrafluorophthalocyanine) Phthalocyanine bis(dimethylhexylvinylsilyloxide)

A mixture of silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(tetrafluorophthalocyanine) phthalocyanine dihydroxide (10.5 mg),7-oct-1-enyl dimethylchlorosilane (38 μL), imidazole (10 mg) anddimethylformamide (200 μL) was stirred at room temperature for 30minutes. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed (silica gel 70-230 mesh, 60Å, 2×50 cm, hexane-toluene), vacuum dried and weighed (4 mg).

UV-vis(tetrahydrofuran) (λ_(max) (nm)): 732, 757, 794, 816.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 763, 830.

EXAMPLE 51 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(tetrafluorophthalocyanine) Phthalocyanine bis(dimethylpentafluorophenylsilyloxide)

A mixture of silicon di(1,6-diphenyl-2,3-naphthalocyanine)!(tetrafluorophthalocyanine) phthalocyanine dihydroxide (10.5 mg),chlorodimethylpentafluorophenylsilane (28 μL), imidazole (10 mg) anddimethylformamide (200 μL) was stirred at room temperature for 30minutes. The resultant was concentrated under vacuum on the rotaryevaporator. The residue was chromatographed (silica gel 70-230 mesh, 60Å, 2×50 cm, hexane-toluene) to afford two product fractions A and Bwhich were vacuum dried and weighed (2.8 mg and 5.5 mg, respectively).

A. UV-vis(tetrahydrofuran) (λ_(max) (nm)): 650, 726, 762, 796, 824.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 770.

B. UV-vis(tetrahydrofuran) (λ_(max) (nm)): 651, 726, 763, 796, 824.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 770.

EXAMPLE 52 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!Diphthalocyanine bis(dimethylpentafluorophenylsilyloxide)

A mixture of silicon di(1,6-diphenyl-2,3-naphthalocyanine)!diphthalocyanine dihydroxide (20 mg),chlorodimethylpenta-fluorophenylsilane (58 μL), imidazole (20 mg) anddimethylformamide (450 μL) was stirred at room temperature for 1 hour.The resultant was concentrated under vacuum on the rotary evaporator.The residue was treated with hexane (5 mL) and the green solid productcollected by filtration, washed with hexane (2 mL), vacuum dried andweighed (26 mg).

UV-vis(tetrahydrofuran) (λ_(max) (nm)): 648, 691, 724, 759.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 768.

EXAMPLE 53 Synthesis ofDi(1,6-diphenylnapthalocyanine)di(tert-butylphthalocyanine)

A mixture of 1,4-diphenylnaphthalene dicarbonitrile (495 mg),4-tert-butylphthalonitrile (92 mg), and lithium butoxide (4.0 mL) wasrefluxed in an oil bath for 1.5 hours and cooled. Cold glacial aceticacid (20 mL) was added to the suspension formed and vacuum dried. Thegreen residue was resuspended in dichloromethane and the solutioncentrifuged at 3000 rpm for 15 minutes. The supernatant was washed with1N HCL (2×20mL) followed by water (1×10 mL). The organic layer was driedunder vacuum. The crude product was chromatographed (silica gel 70-230mesh, 60 Å, 2×50 cm, hexane-toluene), vacuum dried, and weighed (4.2mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm), ε(M⁻¹ cm⁻¹)): 668, 43297; 688,86914; 726, 92715; 758, 64329.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 732.

EXAMPLE 54 Synthesis of 5-tert-butyl-1,3-diiminoisolindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of4-tert-butylphthalonitrile (1.8 g), 25% sodium methoxide in methanol(2.3 mL), and dry 1-pentanol (20 mL) for 30 minutes. With continuedammonia introduction, the mixture was refluxed for 1.5 hours. After theresultant had cooled, the solvent was removed with a rotary evaporator.The residue was treated with methylene chloride (20 mL) and the productwas collected by filtration, washed twice with methylene chloride (10 mLeach time), ether (10 mL), vacuum dried and weighed (0.4 g).

EXAMPLE 55 Synthesis of 6,7-Dibromo-1,3-diiminobenz f!isoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of6,7-dibromonaphthalene-2,3-dicarbonitrile (0.5 g), 25% sodium methoxidein methanol (0.3 mL), and dry 1-pentanol (10 mL) for 50 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 2.5 hours.After the resultant had cooled, the orange-yellow solid was collected byfiltration and washed with ether (20 mL), vacuum dried and weighed (0.6g).

EXAMPLE 56 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)Di-tert-butylphthalocyanine! Dihydroxide

Silicon tetrachloride (57 μL) was added to a mixture ofdiphenyl-1,3-diiminobenz f!isoindoline (172 mg) and5-tert-butyl-1,3-diiminoisoindoline (50 mg) in freshly distilledquinoline (1 mL) under an argon atmosphere and the mixture heated withstirring at 210° C. for 1 hour. The resultant was allowed to coolslightly, treated with water (2 mL) and refluxed for 5 minutes. Themixture was cooled, treated with ether (10 mL) and filtered washing thesolid with ether (30 mL). The organic layer of the filtrate wasseparated, washed twice with water (20 mL each time), dried (Na₂ SO₄)and the ether evaporated with a rotary evaporator. The residue waschromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm, methylenechloride), vacuum dried and weighed (11 mg, green solid).

UV-vis (methylene chloride) (λ_(max) (nm)) 656, 670, 694, 730, 758.

Fluorescence (methylene chloride) (λ_(max) (nm)): 767.

EXAMPLE 57 Synthesis of Silicon di(1,6-diphenyl-2,3-naphthalocyanine)!Di-tert-butylphthalocyanine bis(dimethylhexylvinylsilyloxide)

A mixture of silicon di(1,6-diphenyl-2,3-naphthalocyanine) !di(tert-butylphthalocyanine) dihydroxide (320 mg), 7-oct-1-enyldimethylchlorosilane (200 μL), imidazole (136 mg) and dimethylformamide(6 mL) was stirred at room temperature for 12 hours. The resultant wasconcentrated under vacuum on the rotary evaporator. The residue waschromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm, hexane). Theblue fraction was collected, the solvent evaporated with a rotaryevaporator, and weighed (150 mg).

UV-vis (methylene chloride) (λ_(max) (nm)): 632, 676, 702, 750.

Fluorescence (methylene chloride) (λ_(max) (nm)): 716.

EXAMPLE 58 Synthesis of Silicon Octabromo-2,3-naphthalocyanineDihydroxide

Silicon tetrachloride (114 μL) was added to a mixture of6,7-dibromo-1,3-diiminobenz f!isoindoline (433 mg) and5-tert-butyl-1,3-diiminoisoindoline (100 mg) in freshly distilledquinoline (2 mL) under an argon atmosphere and the mixture heated withstirring at 210° C. for 2 hours. The resultant was allowed to coolslightly, treated with water (2 mL) and refluxed for 15 minutes. Themixture was cooled, treated with ether (4 mL) and filtered washing thesolid twice with ether (2 mL each time). The solid was vacuum dried andweighed (0.57 g, dark green solid).

EXAMPLE 59 Synthesis of Silicon Octabromo-2,3-naphthalocyaninebis(dimethylhexylvinylsilyloxide)

A mixture of silicon octabromo-2,3-naphthalocyanine dihydroxide (500mg), 7-oct-1-enyl dimethylchlorosilane (256 μL), imidazole (68 mg) anddimethylformamide (5 mL) was stirred at room temperature for 12 hours.The resultant was concentrated under vacuum with a rotary evaporator.The residue was chromatographed (silica gel 70-230 mesh, 60 Å, 2×50 cm,hexane), the blue-green fraction collected, vacuum dried, and weighed(300 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 694.

Fluorescence (tetrahydrofuran) (λ_(max) (nm)): 706.

EXAMPLE 60 Synthesis of Silicon Octaethoxyphthalocyanine Dichloride

Silicon tetrachloride (600 μL) was added to a mixture of4,7-diethoxy-1,3-diiminoisoindoline (1.0 g) in freshly distilledquinoline (10 mL) under an argon atmosphere and the mixture heated withstirring at 200° C. for 1.5 hours. The resultant was allowed to cool andtreated with water (10 mL) and methylene chloride (10 mL). The organiclayer was separated and evaporated with a rotary evaporator. The blackresidue was treated with ether (5 mL) and filtered. The filtrate wasdried (Na₂ SO₄) and the solvent evaporated with a rotary evaporator,vacuum dried and weighed(300 mg, dark green solid).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 742.

UV-vis (methylene chloride) (λ_(max) (nm)): 764.

Infrared spectrum (KBr): 3435, 3060, 2983, 2932, 2228, 1727, 1603, 1504,1317, 1256, 1218, 1068, 810 cm⁻¹.

EXAMPLE 61 Synthesis of Diethoxy-1,3-diiminoisoindoline

Anhydrous ammonia was slowly bubbled through a stirred mixture of1,4-diethoxy-2,3-phthalonitrile (1.0 g), 25% sodium methoxide inmethanol (1.2 mL), and dry 1-pentanol (20 mL) for 45 minutes. Withcontinued ammonia introduction, the mixture was refluxed for 3 hours.After the resultant had cooled, the solvent was removed with a rotaryevaporator. The residue was dried under vacuum and weighed (1.4 g, greensolid).

EXAMPLE 62 Synthesis of Octamethoxy-2,3-naphthalocyanine

1,4-dimethoxynaphthalene-2,3-dicarbonitrile (820 mg) suspended in 25%sodium methoxide in methanol (7 mL) was refluxed for 1.5 hours, cooled,and stirred into glacial acetic acid (50 mL). After 30 minutes, thesolvent was evaporated with a rotary evaporator and the residuedissolved in methylene chloride (100 mL). The solution was washed with10% hydrochloric acid (100 mL), brine (100 mL) and evaporated with arotary evaporator. The residue was chromatographed (silica gel 70-230mesh, 60 Å, 2×50 cm, toluene), vacuum dried and weighed (52 mg,red-brown solid).

UV-vis (tetrahydrofuran) (λ_(max) (nm)): 837.

EXAMPLE 63 Synthesis of Germanium tetra-tert-ButylphthalocyanineDichloride

Germanium tetrachloride (1.5 mL) was added to a mixture of5-tert-butyl-1,3-diiminoisoindoline (500 mg) and tributylamine(3.4 mL)in 1,2,3,4-tetrahydronaphthalene (7 mL) under an argon atmosphere andthe mixture refluxed for 3.5 hours. The resultant was allowed to cool,treated with water (20 mL) and methylene chloride (20 mL). The organiclayer was separated, washed with water (10 mL), dried (MgSO₄) andevaporated with a rotary evaporator. The residue was chromatographed(silica gel 70-230 mesh, 60 Å, 2×50 cm, toluene:isopropanol (9:1)), thegreen fraction collected, vacuum dried, and weighed (310 mg).

UV-vis (tetrahydrofuran) (λ_(max) (nm)) 680.

Fluorescence(tetrahydrofuran) (λ_(max) (nm)): 718, 750.

EXAMPLE 64 Effect of Human Serum on the Fluorescence Intensities ofVarious Dye Systems in Latex with Different Stokes Shifts and Excitationand Emission Wavelengths

Donor and acceptor dye pairs or a hybrid phthalocyanine derivative aslisted in Table 3 were incorporated into 0.2 micron latex (CML from IDC,Portland, Oreg.) using the tetrahydrofuran solvent method. The latexparticles were diluted to various solids concentrations as indicated inthe Table into either a buffer containing 50 mM potassium phosphate, 10mM potassium borate, 150 mM sodium chloride and 10 mg/mL bovine serumalbumin, pH 7 or neat human serum. The excitation and emissionwavelengths and the corresponding Stokes shift are as indicated in theTable.

The results show that the fluorescence intensities measured in neathuman serum are greatly affected when the excitation wavelength is in aregion where human serum absorbs. Conversely, the fluorescenceintensities of latex measured in human serum are not affected when theexcitation wavelength is in the region where the serum does notsignificantly absorb.

                                      TABLE 3                                     __________________________________________________________________________                               Fluores-                                                                           Latex                                         Dye System   Excitation                                                                         Emission                                                                           Stokes                                                                            cence                                                                              Solids                                        (Donor/Acceptor)                                                                           (nm) (nm) Shift                                                                             Intensity*                                                                         (%)                                           __________________________________________________________________________    trans-4- 4-(Dibutylamino)                                                                  475  680  205                                                    styryl!-1-methyl pyridinium                                                   Iodide/Silicon phthalo-                                                       cyanine bis(dimethylvinyl-                                                    silyloxide)                                                                   Buffer                     369.0                                                                              0.0019                                        Serum                      28.0 0.0019                                        Meso-tetra-2-aminophenyl                                                                   420  680  260                                                    porphine/Silicon phthalo-                                                     cyanine bis(dimethylvinyl-                                                    silyloxide)                                                                   Buffer                     257.0                                                                              0.0010                                        Serum                      72.0 0.0010                                        (E,E)-3,5-bis-(4-phenyl-1,3-                                                               670  780  110                                                    butadienyl)-4,4-difluoro-4-                                                   bora-3a,4a-diazo-s-                                                           indacene/Silicon 2,3-                                                         naphthalocyanine bis                                                          (dimethylhexylvinylsilyl-                                                     oxide)                                                                        Buffer                     20.6 0.0005                                        Serum                      19.5 0.0005                                        1,1'-Dihexyl-3,3,3',3'-                                                                    650  780  130                                                    tetramethylindodicarbo-                                                       cyanine Iodide/Silicon 2,3-                                                   naphthalocyanine bis                                                          (dimethylhexylvinylsilyl-                                                     oxide)                                                                        Buffer                     28.9 0.0005                                        Serum                      30.2 0.0005                                        Hybrid Compound                                                               Silicon  di(1,6-diphenyl-                                                                  646  760  114                                                    naphthalocyanine)!                                                            diphthalocyanine bis                                                          (dimethylhexylvinylsilyl-                                                     oxide)                                                                        Buffer                     49.7 0.0007                                        Serum                      45.3 0.0007                                        __________________________________________________________________________     *The fluorescence intensities are not corrected.                         

EXAMPLE 65 Effect of Axial Ligand on the Quenching of Silicondi(1,6-diphenylnaphthalocyanine)!diphthalocyanines

Silicon di(1,6-diphenylnaphthalocyanine)! diphthalocyanine dihydroxideand Silicon di(1,6-diphenylnaphthalocyanine)! diphthalocyanine bisdimethylhexylvinylsilyloxide! were incorporated into 0.2 micron CMLlatex (IDC Corporation, Portland Oreg.) at various dye concentrations asindicated in the Table below using the THF solvent system. Thefluorescent latexes were diluted to 0.00057% solids in either 5 mMpotassium phosphate, 1 mM potassium borate buffer, pH 7 or intetrahydrofuran. The fluorescence intensities were measured byexcitation at 646 nm. Emission was set at 760 nm. The results arepresented below in Table 4.

The results show that the dihydroxy hybrid derivative, which has noaxial ligand, has a large degree of quenching, even at 0.1 mg/mL dyeloading while the bis dimethylhexylvinylsilyloxide hybrid derivative(with the axial ligand) has very little quenching. The results indicatethat axial ligands are important for hybrid phthalocyanine derivativesto attain maximum fluorescence intensities in particles.

                                      TABLE 4                                     __________________________________________________________________________                  Fluorescence                                                                         Percent Quench                                                  Percent                                                                              Intensity of                                                                         of Silicon                                                                           Fluorescence                                             Quench of                                                                            Latex con-                                                                            di(1,6-                                                                             Intensity of Latex                                       Silicon                                                                              taining Silicon                                                                      diphenyl-                                                                            containing Silicon                                        di(1,6-                                                                              di(1,6-                                                                             naphthalo-                                                                            di(1,6-diphenyl-                                        diphenyl-                                                                            diphenyl-                                                                            cyanine)!                                                                            naphthalo-                                               naphthalo-                                                                           naphthalo-                                                                           diphthalo-                                                                           cyanine)!                                         Concentration                                                                        cyanine)!                                                                            cyanine)!                                                                            cyanine bis                                                                          diphthalo-                                        of dye per mL                                                                        diphthalo-                                                                           diphthalo-                                                                            dimethyl-                                                                           cyanine bis                                       of 2% solid                                                                          cyanine                                                                              cyanine                                                                              hexylvinylsilyl-                                                                      dimethylhexyl-                                   (mg)   dihydroxide                                                                          dihydroxide                                                                          oxidel vinylsilyloxide!                                  __________________________________________________________________________    0.1    89     1      0      4                                                 0.2    75     2      6      7                                                 0.3    80     2      0      10                                                0.4    78     3      2      13                                                0.6    82     2      3      16                                                0.8    84     1      5      19                                                __________________________________________________________________________

EXAMPLE 66 Comparison of Quenching in Latex for a Hybrid PhthalocyanineDerivative and a Naphthalocyanine Derivative, Both with Axial Ligands

Silicon di(1,6-diphenylnaphthaolcyanine)! diphthalocyanine bisdimethylhexylvinylsilyloxide! (hybrid phthalocyanine derivative) andsilicon 2,3-naphthalocyanine bis dimethylhexylvinylsilyloxide!(naphthalocyanine derivative) were incorporated into 0.2 micron CMLlatex (IDC Corporation, Portland Oreg.) at various dye concentrations asindicated in the Table below using the tetrahydrofuran solvent system.The fluorescent latexes were diluted to 0.00057% solids in either 5 mMpotassium phosphate, 1 mM potassium borate buffer, pH 7 or intetrahydrofuran. The fluorescence intensities were measured atexcitation and emission wavelengths as indicated in the Table below.

The results show that the hybrid phthalocyanine derivative is much moreresistant to quenching than the naphthalocyanine derivative. The resultsshow the special properties of the hybrid phthalocyanine derivatives forattaining improved fluorescence intensities in latex.

                  TABLE 5                                                         ______________________________________                                        Silicon 2,3-         Percent          Percent                                 naphthalocyanine                                                                         Fluorescence                                                                            Quench   Fluorescence                                                                          Quench                                  bis(dimethylhexyl-                                                                       Intensity of                                                                            (Ex. 350 Intensity of                                                                          (Ex. 650                                vinylsilyloxide)                                                                         Latex     nm       Latex   nm                                      concentration                                                                            (Ex. 350 nm                                                                             Em. 780  (Ex. 650 nm                                                                           Em 780                                  mg/mL      Em. 780 nm)                                                                             nm)      Em. 780 nm)                                                                           nm)                                     ______________________________________                                        0.1        11        0        1       15                                      0.3        34        13       3       30                                      0.5        41        19       4       34                                      0.7        63        26       6       41                                      0.9        31        32       3       46                                      1.0        31        28       3       42                                      2.0        33        36       3       47                                      ______________________________________                                        Silicon  di(1,6-                                                              diphenylnaphtha-                                                              locyanine)!          Percent          Percent                                 diphthalocyanine                                                                         Fluorescence                                                                            Quench   Fluorescence                                                                          Quench                                  bis dimethyl-                                                                            Intensity of                                                                            (Ex. 350 Intensity of                                                                          (Ex. 650                                hexylvinylsilyl-                                                                         Latex     nm       Latex   nm                                      oxide) concentra-                                                                        (Ex. 350 nm                                                                             Em. 760  (Ex. 650 nm                                                                           Em 760                                  tion (mg/mL)                                                                             Em. 760 nm)                                                                             nm)      Em. 760 nm)                                                                           nm)                                     ______________________________________                                        0.1        11        0        6       0                                       0.3        31        0        16      0                                       0.5        56        0        28      0                                       0.7        60        0        30      0                                       0.9        78        0        39      0                                       1.0        82        0        41      0                                       2.0        113       0        58      13                                      ______________________________________                                    

We claim:
 1. A composition made by a process comprising steps of:A)selecting at least one fluorescent hybrid phthalocyanine derivativecontaining a metal, said derivative(s) having (1) at least one donorsubunit with a desired excitation peak; and (2) at least one acceptorsubunit with a desired emission peak; and (3) at least one electrontransfer subunit; and (4) at least one axial ligand covalently bound tosaid metal in said phthalocyanine derivative; wherein said derivative(s)is/are capable of intramolecular energy transfer from said donor subunitto said acceptor subunit; and, B) randomly incorporating saidphthalocyanine derivative(s) into a microparticle selected from thegroup consisting of latex, silica, alumina, liposomes and colloids.
 2. Aloadable microparticle comprising a fluorescent phthalocyaninederivative having a metal with at least one axial ligand covalentlybound to said metal in said phthalocyanine derivative.
 3. A loadablemicroparticle comprising a fluorescent hybrid phthalocyanine derivativehaving a metal with at least one axial ligand covalently bound to saidmetal in said hybrid phthalocyanine derivative.
 4. A fluorescentmicroparticle made by the process comprising: A) selecting a series offluorescent dyes comprising at least one initial donor dye with adesired excitation peak of greater than approximately 500 nm and two ormore final acceptor dyes with desired emission peaks equal to or verysimilar to each other, wherein each dye in the series has a spectraloverlap sufficient to allow for significant energy transfer ofexcitation energy to the final acceptor dyes and wherein a Stokes shiftof treater than or equal to 50 nm exists between the energy donor and atleast one of the energy acceptors, and at least one of said acceptordyes has an emission wavelength greater than approximately 680 nm; andB) randomly incorporating said series of dyes in a microparticle,whereby the improved particle exhibits minimal fluorescence quenchingand maximum fluorescence intensity.
 5. The microparticle of claim 4wherein said emission peaks are within 10 nm of each other.